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21EBTA Driving Pluripotent Stem Cell Osteogenesis with Light for Tissue Engineering

Reference	BB/W013940/1
Principal Investigator / Supervisor	Professor Susan Kimber
Co-Investigators / Co-Supervisors	Dr Marco Andre Neves Domingos
Institution	The University of Manchester
Department	School of Biological Sciences
Funding type	Research
Value (£)	360,227
Status	Current
Type	Research Grant
Start date	31/01/2022
End date	30/01/2024
Duration	24 months

Abstract

Skeletal disease affecting bone and cartilage such as non-union fractures or osteoarthritis are a major cause of morbidity and disability in the over 45s. We will generate bone constructs from engineered stem cells using visible light. While some growth factors important for bone development such as Wnts can now be substituted by small molecules (e.g.CHIR-99021- GSK-3Beta inhibitor) there is no similar substitute for costly bone morphogenetic proteins (BMPs). We will use optogenetics to engineer human pluripotent stem cells (hPSCs) through Crispr/Cas9 gene editing, to express BMP receptors (BMPR1A and BMPRII) sensitive to visible light, and evaluate light response using a custom LED device. By employing engineered receptors with narrow red light wavelength sensitivity, we will have exquisite control of cellular signalling responses in both space and time. This will also allow far greater control of signalling intensity than just daily addition of (labile) growth factor, and will remove the need for expensive and batch-variable BMP. We will develop a novel method, applying light driven growth factor signalling to induce the osteogenic differentiation of hPSCs, adapting in house and published protocols for hPSC-osteogenesis. The best protocol will be used to pilot the bioprinting of the osteoprogenitors encapsulated in collagen 1 and hydroxyapatite containing hydrogels, to form 3D tissue engineered, mineralising bone constructs, which we will characterise. We propose to take this forward in future work in 3 ways 1) to engineer a bone constructs suitable for healing bone lesions; 2) to combine with light driven chondrogenic constructs to engineer a combined osteochondral construct for investigation of joint disease. 3) to use in drug discovery for skeletal diseases. This proposal will generate an optogenetic pipe line that can be applied to other stem cell systems dependent on BMPs and indeed used to engineer other light-driven growth factor response systems.

Summary

Skeletal problems affecting bones and joints, such as fractures that don't heal or osteoarthritis are a major cause of disfunction, pain and disability in the over 45s. Human pluripotent stem cell can make different tissues, including bone, if the right reagents are added to the cells in a dish at appropriate times. However, generating such tissues is costly, relying on addition of proteins called growth factors- in particular one called bone morphogenetic protein (BMP). BMPs are important in development for making bone, but the protein is expensive and different batches have different activity, reducing the reproducibility of protocols. We will replace the use of BMP in our protocol by engineering stem cells to contain receptors which respond to a particular wavelength of visible light, instead of the BMP protein, to trigger the effect of BMP on the cells. We will evaluate the cell-response to light, in terms of intensity and timing of light pulses, to see if it replicates what the BMP protein does to the cells. As well as saving costs, this will give us much more accurate control of the bone-encouraging signals, compared to that obtainable by daily addition of the protein which is broken down by the cells. We will thus develop a novel method, driving the stem cells to form bone using light. First this will be done in a dish but then we will transfer this to a 3 dimensional format; we will use a 3D printer to print the light responsive cells to make a 3D construct by incorporating them into a gel containing collagen, found in bone, and hydroxyapatite which will encourage mineralisation of the bone. We will combine this with a stiffer scaffold to encourage bone formation. We will monitor the printing and culture parameters to give the most authentic bone tissue and characterise this. Having established this system, in the future we will take this forward in 3 ways 1) to make bone-like constructs suitable for healing bone lesions; 2) to combine with light driven, pluripotent stem cell-generated, cartilage cells and make a construct which can be used to investigate the formation of the joint and factors causing joint disease; 3) to use as a model system for discovering new drugs which enhance bone healing or correct bone or joint disease abnormalities. These light-driven engineered cells can also be applied to other BMP-dependent human stem cell generated tissues, for understanding development and disease, and the pipeline can be used to engineer other growth factor responses applicable to human health and manufacturing processes. Thus this marks some of the first step in this promising area of synthetic biology, opening up the use of light with engineered human pluripotent stem cells and bioprinting to drive different applications.

Committee	Not funded via Committee
Research Topics	Synthetic Biology, Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	Engineering Biology Transition Awards [2021]
Funding Scheme	X – not Funded via a specific Funding Scheme

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