Periodic Reporting for period 2 - PreciseCellPD (A PRECISION CELL REPLACEMENT STRATEGY FOR PARKINSON’S DISEASE)
Reporting period: 2022-09-01 to 2024-02-29
Parkinson’s disease (PD) is an incurable neurodegenerative disorder clinically characterized by bradykinesia, rigidity, resting tremor, gait disturbances and postural instability, as well as non-motor features. The main motor features of the disease are associated with the loss of midbrain dopaminergic (mDA) neurons of the substantia nigra pars compacta (A9/SNc) and their projection to the striatum, especially the putamen.
Proof of concept clinical studies have demonstrated that it is possible to replace cells lost to disease by transplanting foetal midbrain tissue cell suspensions containing mDA progenitors. More recently human pluripotent stem cells (hPSCs) have been used to generate mDA progenitors, which have been successfully used for transplantation in animal models of PD. However we recently found that current hPSC-derived progenitor cell preparations contain a very large repertoire of cell types, several of which are either though to be irrelevant or even potentially deleterious for the purpose of cell replacement.
PD is the second most common neurodegenerative disorder and it is becoming more prevalent as the age of our population increases. Current treatments for PD focus on symptomatic management through pharmacological restoration of dopaminergic activity with drugs such as L-DOPA. However, these treatments do not change the progressive degeneration or the course of the disease, and over time they give side effects, such as motor fluctuations, dyskinesias and neuropsychiatric problems. It is currently thought that targeted therapies, such as replacing nigral A9 neurons could dramatically change the natural history of treated PD and alleviate the health, social and economical burden caused by this disease in our society.
Our objective of the project is to develop a precision cell replacement therapy for PD in which only the cell-types required for therapeutic effect are transplanted.
To achieve this we first decided to address two critical knowledge gaps: (i) Define the molecular diversity of adult human mDA neurons at the single-cell level, and (ii) Understand the mechanisms controlling human A9/SNc neuron specification and maintenance. Second, with this information, we aim at developing two novel alternative regenerative medicine approaches based on the transplantation of hPSC-derived cell types or the in vivo reprogramming of brain cells in rodent models of PD.
Proof of concept clinical studies have demonstrated that it is possible to replace cells lost to disease by transplanting foetal midbrain tissue cell suspensions containing mDA progenitors. More recently human pluripotent stem cells (hPSCs) have been used to generate mDA progenitors, which have been successfully used for transplantation in animal models of PD. However we recently found that current hPSC-derived progenitor cell preparations contain a very large repertoire of cell types, several of which are either though to be irrelevant or even potentially deleterious for the purpose of cell replacement.
PD is the second most common neurodegenerative disorder and it is becoming more prevalent as the age of our population increases. Current treatments for PD focus on symptomatic management through pharmacological restoration of dopaminergic activity with drugs such as L-DOPA. However, these treatments do not change the progressive degeneration or the course of the disease, and over time they give side effects, such as motor fluctuations, dyskinesias and neuropsychiatric problems. It is currently thought that targeted therapies, such as replacing nigral A9 neurons could dramatically change the natural history of treated PD and alleviate the health, social and economical burden caused by this disease in our society.
Our objective of the project is to develop a precision cell replacement therapy for PD in which only the cell-types required for therapeutic effect are transplanted.
To achieve this we first decided to address two critical knowledge gaps: (i) Define the molecular diversity of adult human mDA neurons at the single-cell level, and (ii) Understand the mechanisms controlling human A9/SNc neuron specification and maintenance. Second, with this information, we aim at developing two novel alternative regenerative medicine approaches based on the transplantation of hPSC-derived cell types or the in vivo reprogramming of brain cells in rodent models of PD.
The project has advanced in a satisfactory manner towards our three main objectives:
1) Define adult human mDA neuron subtypes, including A9/SNc neurons. In this area we have analyzed scRNA-seq data from adult substantia nigra samples and identified different subtypes of dopaminergic neurons (aim 1.1). A manuscript has been recently submitted (Siletti et al. bioRxiv; https://doi.org/10.1101/2022.10.12.511898). We have also started to test candidate factors for their capacity to differenttiate mDA progenitors into DA neuron subtypes.
2) Develop a cell-replacement strategy for PD based on the transplantation of hPSC-derived mDA progenitors differentiating into A9/SNc neurons. Here we have made progress in two directions: (i) We have generated vectors to make stable inducible CRISPR hPSC lines for KO, repression and activation. CRISPR cell lines are currently being characterized. (ii) We have further improved our protocol for the differentiation of hESCs into molecularly defined midbrain cell types. A manuscript has been recently accepted for publication (Nishimura et al., 2022, Stem cell reports; https://doi.org/10.1101/2022.09.15.507987) and a second manuscript has been submitted (Pantazis et al., bioRxiv; https://doi.org/10.1101/2021.12.15.472643).
3) Develop a novel cell-replacement strategy for PD based on the in situ conversion of striatal astrocytes into induced A9/SNc mDA neurons by direct in vivo reprogramming. In this section, we are currently exploring new factors for their capacity to convert human astrocytes into mDA neuron subtypes in vitro.
1) Define adult human mDA neuron subtypes, including A9/SNc neurons. In this area we have analyzed scRNA-seq data from adult substantia nigra samples and identified different subtypes of dopaminergic neurons (aim 1.1). A manuscript has been recently submitted (Siletti et al. bioRxiv; https://doi.org/10.1101/2022.10.12.511898). We have also started to test candidate factors for their capacity to differenttiate mDA progenitors into DA neuron subtypes.
2) Develop a cell-replacement strategy for PD based on the transplantation of hPSC-derived mDA progenitors differentiating into A9/SNc neurons. Here we have made progress in two directions: (i) We have generated vectors to make stable inducible CRISPR hPSC lines for KO, repression and activation. CRISPR cell lines are currently being characterized. (ii) We have further improved our protocol for the differentiation of hESCs into molecularly defined midbrain cell types. A manuscript has been recently accepted for publication (Nishimura et al., 2022, Stem cell reports; https://doi.org/10.1101/2022.09.15.507987) and a second manuscript has been submitted (Pantazis et al., bioRxiv; https://doi.org/10.1101/2021.12.15.472643).
3) Develop a novel cell-replacement strategy for PD based on the in situ conversion of striatal astrocytes into induced A9/SNc mDA neurons by direct in vivo reprogramming. In this section, we are currently exploring new factors for their capacity to convert human astrocytes into mDA neuron subtypes in vitro.
Our work has uncovered the molecular diversity of dopaminergic neurons in the adult human midbrain, which was the first main challenge and knowledge gap during the first reporting period of the project. In the reminder of the project we expect to contribute ground-breaking knowledge of the molecular mechanisms that control the identity and maintenance of human A9/SNc dopaminergic neurons. All this knowledge will be used to develop and improve two novel cell-replacement strategies in rodent models of PD. We expect this project will pave the way for precision cell-replacement therapies for PD and will lead to a paradigm shift in which the focus will change from transplanting a full tissue to replacing only the specific cell type/s required to restore the functions lost to PD.