OPTOGENETIC CONTROL OF BMP SIGNALLING TO DRIVE HPSC CHONDROGENESIS

Abstract

Regenerative medicine approaches for the treatment of osteoarthritis have the potential to provide a long-term therapeutic solution to a debilitating and painful disease. Human pluripotent stem cells (hPSCs) have been suggested as a potential source for tissue engineering applications due to their advantageous properties of self-renewal and pluripotency. Through applying developmental knowledge, hPSCs can be precisely directed through transitional lineages to generate large numbers of functional chondrocyte progenitor cells. However, efficient generation of cells that exhibit the desired phenotypic characteristics remains a challenge. HPSCs are stimulated with uniform addition of molecules that activate cell signalling pathways to influence direction of differentiation. However, such approaches are limited by batch-to-batch variability in bioactivity and the inability to recapitulate the dynamic nature of developmental morphogenesis. The emergence of optogenetic technologies has led to the development of light-sensitive tools that have been used to control a diverse range of cell-signalling events. Optogenetic approaches enable defined manipulation through combining cell-signalling effectors with light-sensitive molecules, whose activity can then be directed with spatiotemporal precision. Considering the importance of the bone morphogenetic protein (BMP) signalling pathway in chondrogenesis, this project aimed to develop an optogenetic BMP system to drive chondrogenic differentiation of hPSCs. The optogenetic BMP (optoBMP) system consisted of the intracellular regions of BMPR1B/ALK6 or BMPR2 chimerised to a myristoylation signal at the N-terminus for membrane anchorage and the light-sensitive light oxygen voltage (LOV) sensing domain at the C-terminus for light-induced dimerisation. Optogenetic constructs were inserted into a lentiviral doxycycline inducible vector for transgenic integration and expression. Optogenetic activation of canonical BMP signalling was analysed through detection of nuclear P-SMAD1/5, activation of a SMAD1/5/8 transcriptional reporter (BRE) and upregulation of relevant BMP-target gene expression. Human embryonic stem cells (hESCs) were differentiated using a chondrogenic differentiation protocol developed by our research group. BMP growth factors were substituted for an optimised regime of blue light stimulation. Optogenetic receptor transgenes were successfully generated and integrated into SW1353, HEK293T, TC28a2, and MAN13 human embryonic stem cells. Expression of the optoBMP system was regulated by the addition of doxycycline. Blue light illumination of optogenetic cell lines induced phosphorylation and nuclear accumulation of SMAD1/5, indicating optogenetic activation of the canonical BMP pathway. SMAD1/5/8 transcriptional activity was activated by light stimulation, demonstrated through BRE activation and upregulation of BMP-like gene expression. Optogenetic stimulation during hPSC chondrogenic differentiation indicated optoBMP activation could substitute for BMP growth factors and drive differentiation. Furthermore, analyses indicated that optogenetic stimulation initiated a divergent signalling response to that of BMP2 growth factor stimulation, which then seemed to facilitate significantly higher upregulation of key chondrogenic genes, including COL2A1 and SOX9. Findings documented here demonstrate that optogenetics can be utilised to control the BMP signalling pathway and has illustrated the potential for manipulation of chondrogenic processes during hPSC differentiation.

Date of Award	1 Aug 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Susan Kimber (Supervisor) & Robert Lucas (Supervisor)