Periodic Reporting for period 3 - CellFateTech (Biotechnology for investigating cell fate choice)
Periodo di rendicontazione: 2021-04-01 al 2022-11-30
The transition from a stem cell to differentiated progeny is a highly controlled process. However, how differentiation is regulated is not well understood, despite the importance for stem cell biology, cancer biology and regenerative medicine. This comprehension gap exists partly because we lack the tools and formalism to quantify changes in cell state. Furthermore, cell fate decisions have primarily been studied from a biochemical perspective, while mechanical aspects, despite their importance, have been largely overlooked. My laboratory is focused on developing technology platforms based in microfabrication, hydrogels, and microfluidics to illuminate the physical biology of cell fate choice. In order to achieve insight into biological transitions and how they are regulated, a truly multidisciplinary approach must be embraced. Regulation of stem cell behaviour and function is essential for guided stem cell therapies, which could be used to treat diseases and loss of regenerative capacity in ageing organs.
The objectives of the project were as follows:
1. To develop technologies to illuminate transitions underlying cell fate choices. We will combine cell encapsulation and microfluidic devices for cell interrogation,thus enabling the investigation of transitions as a function of time and signalling.
2. To develop StemBond hydrogel substrates, which uniquely possess the capacity for simultaneous control over the mechanical environment and how cells adhere to it, to better control stem cell function.
3. To develop devices to apply active, defined mechanical cues to stem cell cultures to investigate how forces on cells affect their function.
The objectives of the project were as follows:
1. To develop technologies to illuminate transitions underlying cell fate choices. We will combine cell encapsulation and microfluidic devices for cell interrogation,thus enabling the investigation of transitions as a function of time and signalling.
2. To develop StemBond hydrogel substrates, which uniquely possess the capacity for simultaneous control over the mechanical environment and how cells adhere to it, to better control stem cell function.
3. To develop devices to apply active, defined mechanical cues to stem cell cultures to investigate how forces on cells affect their function.
1. We developed a microfluidic platform to interrogate cell state. The platform consists of 16 channels, over each we have independent control. We optimised the cell encapsulation so that we have 3D control over the microenvironment for each cell inserted into the microfluidic device. We have now published this results, in Lab on a Chip (Mulas, Hodson, et al, Lab on a Chip, 2020).
2. One of the challenges we faced in our research so far was the unexpected problem of dealing with the immense amount of technical noise intrinsic to single cell RNA sequencing, which made our measurements of network entropy so far impossible. However, we have now developed models to account for the technical noise, and estimate uncertainty. These models are novel, and we believe they will be of great interest to the computational biology community. We are in the process of writing them up. Once we do, we will put it on a preprint server and begin the process of speaking to other experts in the field about how our new models can help others better understand the meaning of the data in this important and essential methodology.
3. In the course of our work developing the microfluidic platform to study stem cell fate transitions, we identified a signal that was essential for moving from one stem cell state to another. We performed a mechanistic study to determine exactly how that signal worked on the signalling pathways in stem cells to achieve this transition. This work is currently on a preprint server and under review at Review Commons (Mulas, et al, Biorxiv, 2023).
4.We developed a high-throughput cell stretcher, and were able to use it to show that embryonic stem (ES) cells are not responsive to mechanical cues, and only become so when they begin differentiating. We adapted the cell stretcher to perform calcium imaging and get a real-time readout of signalling as we apply active forces. This is now published in Open Biology (Verstreken, et al, Open Biology, 2019). We also adapted this tissue stretcher to collaborate with a group that used it to show that stretching oesophagus tissue leads to a change in stem cell activity during development (McGinn, et al, Nature Cell Biology, 2021).
5. We made significant progress on optimising and further developing the StemBond hydrogels. We used atomic force microscopy to disentangle stiffness from adhesion, and now understand how these hydrogels offer a significant improvement on previously published cell substrates. Specifically, we showed they are an improvement because the extracellular matrix binding on these gels is more stable than previous protocols, enabling far better and more stable engagement between cells and extracellular matrix (Labouesse, et al, Nature Communications, 2021). We also pushed it in a number of new directions. We published seminal work on the mechanisms of stem cell ageing using StemBond (Segel, et al, Nature, 2019). We are also making significant progress identifying the intracellular signalling pathways involved in cell fate transitions that are affected by mechanical signalling and how mechanics in turn affects cell fate choices in development and stem cells (de Belly, et al, Cell Stem Cell, 2021 and Yanagida, et al, Cell, 2022). We identified an important cross-talk between cell surface mechanics, which regulates signalling that instructs cell fate choice, and the mechanical environment. We have a European and American patent pending on StemBond, and once awarded are pushing it towards an international patent application. StemBond hydrogels have also formed the technology backbone of a new company called StemBond Therapeutics of which I am a cofounder, which is devoted to using mechanical control to better facilitate control of immune response in tissue.
2. One of the challenges we faced in our research so far was the unexpected problem of dealing with the immense amount of technical noise intrinsic to single cell RNA sequencing, which made our measurements of network entropy so far impossible. However, we have now developed models to account for the technical noise, and estimate uncertainty. These models are novel, and we believe they will be of great interest to the computational biology community. We are in the process of writing them up. Once we do, we will put it on a preprint server and begin the process of speaking to other experts in the field about how our new models can help others better understand the meaning of the data in this important and essential methodology.
3. In the course of our work developing the microfluidic platform to study stem cell fate transitions, we identified a signal that was essential for moving from one stem cell state to another. We performed a mechanistic study to determine exactly how that signal worked on the signalling pathways in stem cells to achieve this transition. This work is currently on a preprint server and under review at Review Commons (Mulas, et al, Biorxiv, 2023).
4.We developed a high-throughput cell stretcher, and were able to use it to show that embryonic stem (ES) cells are not responsive to mechanical cues, and only become so when they begin differentiating. We adapted the cell stretcher to perform calcium imaging and get a real-time readout of signalling as we apply active forces. This is now published in Open Biology (Verstreken, et al, Open Biology, 2019). We also adapted this tissue stretcher to collaborate with a group that used it to show that stretching oesophagus tissue leads to a change in stem cell activity during development (McGinn, et al, Nature Cell Biology, 2021).
5. We made significant progress on optimising and further developing the StemBond hydrogels. We used atomic force microscopy to disentangle stiffness from adhesion, and now understand how these hydrogels offer a significant improvement on previously published cell substrates. Specifically, we showed they are an improvement because the extracellular matrix binding on these gels is more stable than previous protocols, enabling far better and more stable engagement between cells and extracellular matrix (Labouesse, et al, Nature Communications, 2021). We also pushed it in a number of new directions. We published seminal work on the mechanisms of stem cell ageing using StemBond (Segel, et al, Nature, 2019). We are also making significant progress identifying the intracellular signalling pathways involved in cell fate transitions that are affected by mechanical signalling and how mechanics in turn affects cell fate choices in development and stem cells (de Belly, et al, Cell Stem Cell, 2021 and Yanagida, et al, Cell, 2022). We identified an important cross-talk between cell surface mechanics, which regulates signalling that instructs cell fate choice, and the mechanical environment. We have a European and American patent pending on StemBond, and once awarded are pushing it towards an international patent application. StemBond hydrogels have also formed the technology backbone of a new company called StemBond Therapeutics of which I am a cofounder, which is devoted to using mechanical control to better facilitate control of immune response in tissue.
The work that has the highest promise of progressing beyond the state-of-the-art is our ongoing work with the StemBond hydrogels. We have identified the stability of the extracellular matrix on these hydrogels as a previously unknown essential variable in stem cell function. The reason for this is that the stability of the ECM greatly affects trafficking of signals from the extracellular environment into stem cells, and thus greatly affects several important signalling pathways. We will tie this to the importance of the mechanical microenvironment in the process of ageing, and associated loss of regenerative capacity in stem cells. These hydrogels have formed the basis of a spinout, StemBond Therapeutics, to employ mechanical control of cell behaviour and function in cell therapy.
With the microfluidics approaches we have been developing, our work on isolating biological noise from the technical noise intrinsic to single cell RNA sequencing will lead to a very important model for the field. With these advances, coupled with our ongoing work to develop reporters to identify tipping points in cell state transitions, we will add significant insight into the regulation of cell state transitions.
With the microfluidics approaches we have been developing, our work on isolating biological noise from the technical noise intrinsic to single cell RNA sequencing will lead to a very important model for the field. With these advances, coupled with our ongoing work to develop reporters to identify tipping points in cell state transitions, we will add significant insight into the regulation of cell state transitions.