Customised Cytokines: A New Paradigm For HSC Fate Engineering
Lead Research Organisation:
UNIVERSITY OF YORK
Abstract
Haematopoietic stem cell transplantation (HSCT) is the most successful cell therapy in clinical use, primarily for the treatment of blood cancers and bone marrow failure syndromes. HSCT carries significant risks, however, including graft-versus-host disease following allogeneic HSCTs and increased risk of relapse due to transplantation of mutated cells in autologous HSCTs. As such, HSCTs are still associated with high morbidity/mortality and have spurred the research community to develop ex vivo HSC expansion methods to increase availability of functional HSCs to meet the demands of successful transplantation, and, more recently, to facilitate corrective gene therapy efforts.
Over the last five years, we have worked closely with international collaborators to significantly improve both mouse and human HSC expansion protocols by understanding the balance of cytokines, employing polymer-based reagents in culture and standardising key reagents. Together, these efforts enable a >200-fold expansion of transplantable HSCs over 28 days1. Despite this remarkable progress, substantial challenges remain since most cells produced are not HSCs, negatively affecting HSC expansion and precluding efficient molecular characterisation and gene editing approaches. Together, this makes balancing the rapid proliferation of HSCs with other cell maturation critical to understand so we can direct it to achieve homogenous populations of functional HSCs.
The cytokines thrombopoietin (TPO) and stem cell factor (SCF) are essential for driving ex vivo HSC expansion. Combining biophysical and functional methods, we have redefined the mechanisms by which TPO activates its receptor, MPL, and recently solved the structure of the TPO-MPL complex, providing, for the first time, detailed information of ligand-receptor interactions2. Using this information, we have engineered a series of TPO modifications (TPOmods) which alter receptor activation, differentially activating downstream signalling and impacting the balance of TPO-driven proliferation and differentiation. Our findings complement previous data showing that SCF modifications can also preferentially improves HSC proliferation while reducing mast cell differentiation. Together, these data support the notion that modified TPO and SCF could be used in combination to vastly improve and standardise HSC expansion cultures without the cost of unwanted differentiation.
In this project, we will address three primary aims. In Aim 1 we will use the high throughput “AVEXIS” method to rapidly screen modified TPO (TPOmods) proteins and characterise their differential growth, signalling and receptor activation. In Aim 2 we will functionally characterise current and novel TPOmods using a combination of mouse and human ex vivo HSC expansion assays and transplantations to fully determine whether TPOmods can improve the number and quality of HSCs in expansion cultures. Aim 3 will combine the most promising TPOmods with a previously described SCF modification (S4-3a) which significantly improves HSC expansion without driving mast cell differentiation. We will determine the signalling interplay between TPOmods and S4-3a, and whether they further improve ex vivo HSC expansion in combination. Overall, this project will deliver novel mechanistic and functional data with the potential to set a new paradigm for using customised cytokines in ex vivo HSC expansion cultures with improved clinical benefit.
Wilkinson, A. C. et al. Nature 571, 117–121 (2019).
Tsutsumi, N. et al. Cell 186, 4189-4203 (2023)
Over the last five years, we have worked closely with international collaborators to significantly improve both mouse and human HSC expansion protocols by understanding the balance of cytokines, employing polymer-based reagents in culture and standardising key reagents. Together, these efforts enable a >200-fold expansion of transplantable HSCs over 28 days1. Despite this remarkable progress, substantial challenges remain since most cells produced are not HSCs, negatively affecting HSC expansion and precluding efficient molecular characterisation and gene editing approaches. Together, this makes balancing the rapid proliferation of HSCs with other cell maturation critical to understand so we can direct it to achieve homogenous populations of functional HSCs.
The cytokines thrombopoietin (TPO) and stem cell factor (SCF) are essential for driving ex vivo HSC expansion. Combining biophysical and functional methods, we have redefined the mechanisms by which TPO activates its receptor, MPL, and recently solved the structure of the TPO-MPL complex, providing, for the first time, detailed information of ligand-receptor interactions2. Using this information, we have engineered a series of TPO modifications (TPOmods) which alter receptor activation, differentially activating downstream signalling and impacting the balance of TPO-driven proliferation and differentiation. Our findings complement previous data showing that SCF modifications can also preferentially improves HSC proliferation while reducing mast cell differentiation. Together, these data support the notion that modified TPO and SCF could be used in combination to vastly improve and standardise HSC expansion cultures without the cost of unwanted differentiation.
In this project, we will address three primary aims. In Aim 1 we will use the high throughput “AVEXIS” method to rapidly screen modified TPO (TPOmods) proteins and characterise their differential growth, signalling and receptor activation. In Aim 2 we will functionally characterise current and novel TPOmods using a combination of mouse and human ex vivo HSC expansion assays and transplantations to fully determine whether TPOmods can improve the number and quality of HSCs in expansion cultures. Aim 3 will combine the most promising TPOmods with a previously described SCF modification (S4-3a) which significantly improves HSC expansion without driving mast cell differentiation. We will determine the signalling interplay between TPOmods and S4-3a, and whether they further improve ex vivo HSC expansion in combination. Overall, this project will deliver novel mechanistic and functional data with the potential to set a new paradigm for using customised cytokines in ex vivo HSC expansion cultures with improved clinical benefit.
Wilkinson, A. C. et al. Nature 571, 117–121 (2019).
Tsutsumi, N. et al. Cell 186, 4189-4203 (2023)
People |
ORCID iD |
| Ian Hitchcock (Principal Investigator) | |
| David Kent (Co-Investigator) |