Background: Stem cell-based therapies show promise as a means of repairing the degenerate intervertebral disc, with growth factors often used alongside cells to help direct differentiation toward a nucleus pulposus (NP)-like phenotype. We previously demonstrated adipose-derived stem cell (ASC) differentiation with GDF6 as optimal for generating NP-like cells through evaluating end-stage differentiation parameters. Here we conducted a time-resolved transcriptomic characterization of ASCs response to GDF6 stimulation to understand the early drivers of differentiation to NP-like cells.
Methods: Human ASCs were treated with recombinant human GDF6 for 2, 6, and 12 h. RNA sequencing and detailed bioinformatic analysis were used to assess differential gene expression, gene ontology (GO), and transcription factor involvement during early differentiation. Quantitative polymerase chain reaction (qPCR) was used to validate RNA sequencing findings and inhibitors used to interrogate Smad and Erk signaling pathways, as well as identify primary and secondary response genes.
Results: The transcriptomic response of ASCs to GDF6 stimulation was time-resolved and highly structured, with "cell differentiation" "developmental processes," and "response to stimulus" identified as key biological process GO terms. The transcription factor ERG1 was identified as a key early response gene. Temporal cluster analysis of differentiation genes identified positive regulation NP cell differentiation, as well as inhibition of osteogenesis and adipogenesis. A role for Smad and Erk signaling in the regulation of GDF6-induced early gene expression response was observed and both primary and secondary response genes were identified.
Conclusions: This study identifies a multifactorial early gene response that contributes to lineage commitment, with the identification of a number of potentially useful early markers of differentiation of ASCs to NP cells. This detailed insight into the molecular processes in response to GDF6 stimulation of ASCs is important for the development of an efficient and efficacious cell-based therapy for intervertebral disc degeneration-associated back pain.
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http://dx.doi.org/10.1002/jsp2.1315 | DOI Listing |
bioRxiv
August 2024
Yale University Medical School, 333 Cedar Street, Room FMB11, New Haven CT 06510.
Rat post-mitotic septal (SEP) neurons, engineered to conditionally proliferate at 33°C, differentiate when arrested at 37.5°C and can be maintained for weeks without cytotoxic effects. Nine independent cDNA libraries were made to follow arrest-induced neural differentiation and innate immune responses in normal (Nl) uninfected and CJ agent infected SEP cells.
View Article and Find Full Text PDFJOR Spine
March 2024
Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health University of Manchester, Manchester Academic Health Sciences Centre Manchester UK.
Background: Stem cell-based therapies show promise as a means of repairing the degenerate intervertebral disc, with growth factors often used alongside cells to help direct differentiation toward a nucleus pulposus (NP)-like phenotype. We previously demonstrated adipose-derived stem cell (ASC) differentiation with GDF6 as optimal for generating NP-like cells through evaluating end-stage differentiation parameters. Here we conducted a time-resolved transcriptomic characterization of ASCs response to GDF6 stimulation to understand the early drivers of differentiation to NP-like cells.
View Article and Find Full Text PDFJOR Spine
March 2023
Raymond Purves Bone and Joint Research Laboratory Kolling Institute, Sydney University Faculty of Medicine and Health, Northern Sydney Area Health District, Royal North Shore Hospital Sydney New South Wales Australia.
The purpose of this review was to evaluate data generated by animal models of intervertebral disc (IVD) degeneration published in the last decade and show how this has made invaluable contributions to the identification of molecular events occurring in and contributing to pain generation. IVD degeneration and associated spinal pain is a complex multifactorial process, its complexity poses difficulties in the selection of the most appropriate therapeutic target to focus on of many potential candidates in the formulation of strategies to alleviate pain perception and to effect disc repair and regeneration and the prevention of associated neuropathic and nociceptive pain. Nerve ingrowth and increased numbers of nociceptors and mechanoreceptors in the degenerate IVD are mechanically stimulated in the biomechanically incompetent abnormally loaded degenerate IVD leading to increased generation of low back pain.
View Article and Find Full Text PDFJ Cell Biochem
October 2022
Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
Hepatic fibrosis can be considered as a deregulated wound healing process in response to chronic liver injury. Bone morphogenetic protein 13 (BMP13) has been described to promote bone and tendon repair. In this study, we aimed to analyze the expression and function of BMP13 in hepatic fibrosis.
View Article and Find Full Text PDFCells
March 2022
Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
Growth differentiation factors (GDFs) regulate homeostasis by amplifying extracellular matrix anabolism and inhibiting pro-inflammatory cytokine production in the intervertebral disc (IVD). The aim of this study was to elucidate the effects of GDF-6 on human IVD nucleus pulposus (NP) cells using a three-dimensional culturing system in vitro and on rat tail IVD tissues using a puncture model in vivo. In vitro, Western blotting showed decreased GDF-6 expression with age and degeneration severity in surgically collected human IVD tissues (n = 12).
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