Periodic Reporting for period 3 - PreciseCellPD (A PRECISION CELL REPLACEMENT STRATEGY FOR PARKINSON’S DISEASE)
Periodo di rendicontazione: 2024-03-01 al 2025-09-30
Parkinsons disease (PD) is the second most common neurodegenerative disorder and it is becoming more prevalent as the age of our population increases. 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 associated with the loss of midbrain dopaminergic (mDA) neurons of the substantia nigra pars compacta (A9/SNpc) and their projection to the striatum, especially the putamen.
As a proof of concept clinical studies have demonstrated that it is possible to replace the cells lost to disease by transplanting foetal midbrain tissue (cell suspensions) containing mDA progenitors. More recently human mDA progenitor derived from pluripotent stem cells (hPSCs) have been generated and successfully grafted in animal models of PD. However we recently found that current hPSC-derived progenitor cell preparations contain a large repertoire of cell types, several of which are either thought to be irrelevant or even potentially deleterious for the purpose of cell replacement.
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 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 was 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 aimed at developing 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.
To conclude, we were able to meet most of our objectives. We published groundbreaking results on human adult as well as developing single-cell analysis of the brain with a detailed roadmap of how human midbrain DA neurons are generated. This was the basis for further investigations on transcription factors and contribution of extracellular modulators important for the development and characterization of mDA neurons as well as for a successful transplantation of hPSC derived progenitor cells.
As a proof of concept clinical studies have demonstrated that it is possible to replace the cells lost to disease by transplanting foetal midbrain tissue (cell suspensions) containing mDA progenitors. More recently human mDA progenitor derived from pluripotent stem cells (hPSCs) have been generated and successfully grafted in animal models of PD. However we recently found that current hPSC-derived progenitor cell preparations contain a large repertoire of cell types, several of which are either thought to be irrelevant or even potentially deleterious for the purpose of cell replacement.
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 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 was 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 aimed at developing 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.
To conclude, we were able to meet most of our objectives. We published groundbreaking results on human adult as well as developing single-cell analysis of the brain with a detailed roadmap of how human midbrain DA neurons are generated. This was the basis for further investigations on transcription factors and contribution of extracellular modulators important for the development and characterization of mDA neurons as well as for a successful transplantation of hPSC derived progenitor cells.
The project advanced in a satisfactory manner towards our three main objectives:
1) Define adult human mDA neuron subtypes, including A9/SNc neurons. We have analyzed scRNA-seq data from adult substantia nigra samples and identified different subtypes of DA neurons (aim 1.1) (Siletti et al. 2023). We found transcriptional profiles ranging from a complete DA phenotype to TH+ neurons with a partial DA and GABAergic phenotypes. In another analysis we analyzed and annotated human embryonic midbrain samples (Braun et al, 2023) and revealed 20 distinct subtypes of TH+ neurons expressing well-known DA markers and unique profiles. This study revealed the generation of DA neurons from midbrain progenitors and the temporal sequence of transcription factors expressed in different cell types along this development. The development of mDA neurons was further investigated and we found Rgl1 to be their neurogenic progenitor (for mDA neurons) and identified the transcription factor BMAL1 as a novel regulator of mDA neurogenesis. (Asgrimsdottir et al, in press, 2026). We progressed in several directions. We characterized the differentiation of hPSCs into mDA neurons at the single cell level and show that differentiation follows key aspects of midbrain development and gives rise to high quality embryonic DA neurons (Nishimura et al, 2023). We also shortened the differentiation protocol and made it more cost-effective (Pantazis et al, 2022). We generated an inducible Cas9 line for a loss of function CRISPR screen to find transcription factor candidates important for lineage specification (FOXA2, LMX1A, EN1, ASCL1, NEUROG2, NR4A2, TH, DDC and SOX6). We also examined the contribution of extracellular modulators of mDA differentiation and show that the heparan sulfate (HS)-modifying enzymes SULF1 and SULF2 are key modulators of anterior-posterior and dorsal-ventral patterning during mDA neuron development in vitro (Tremolanti et al, manuscript under revision)
2) Develop a cell-replacement strategy for PD based on the transplantation of hPSC-derived mDA progenitors differentiating into A9/SNc neurons. In an in silico forward programming simulation we used data from the literature and identified a number of candidates which were also hand-curated in the targeted transcription factor sgRNA library that was used for the loss of function screen in aim 1.
Transplantation of human PSC derived mDA progenitors into hemiparkinsonian immunocompromised mice, which previously have not been utilized in intracranial grafting experiments, restored motor function. Progenitors were shown to mature into a large percentage of DA neurons expressing canonical markers for A9-subtype mDA neurons, which was further confirmed by a comparison of the transcriptional profiles to those of native adult human mDA cells showing a successful generation of in vivo differentiated cells resembling human SOX6 mDA neurons (Garcia Swinburn et al, submitted manuscript).
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. We improved a previous protocol which resulted in a 70% increase of TH + neurons. We also performed single cell RNA sequencing to compare the in vivo reprogrammed mDA neurons to human embryonic mDA neurons. This analysis is under way.
Our work resulted in a total of 6 publications, 1 manuscript in press, 1 manuscript under revision, 1 submitted manuscript, 2 manuscripts in preparation.
1) Define adult human mDA neuron subtypes, including A9/SNc neurons. We have analyzed scRNA-seq data from adult substantia nigra samples and identified different subtypes of DA neurons (aim 1.1) (Siletti et al. 2023). We found transcriptional profiles ranging from a complete DA phenotype to TH+ neurons with a partial DA and GABAergic phenotypes. In another analysis we analyzed and annotated human embryonic midbrain samples (Braun et al, 2023) and revealed 20 distinct subtypes of TH+ neurons expressing well-known DA markers and unique profiles. This study revealed the generation of DA neurons from midbrain progenitors and the temporal sequence of transcription factors expressed in different cell types along this development. The development of mDA neurons was further investigated and we found Rgl1 to be their neurogenic progenitor (for mDA neurons) and identified the transcription factor BMAL1 as a novel regulator of mDA neurogenesis. (Asgrimsdottir et al, in press, 2026). We progressed in several directions. We characterized the differentiation of hPSCs into mDA neurons at the single cell level and show that differentiation follows key aspects of midbrain development and gives rise to high quality embryonic DA neurons (Nishimura et al, 2023). We also shortened the differentiation protocol and made it more cost-effective (Pantazis et al, 2022). We generated an inducible Cas9 line for a loss of function CRISPR screen to find transcription factor candidates important for lineage specification (FOXA2, LMX1A, EN1, ASCL1, NEUROG2, NR4A2, TH, DDC and SOX6). We also examined the contribution of extracellular modulators of mDA differentiation and show that the heparan sulfate (HS)-modifying enzymes SULF1 and SULF2 are key modulators of anterior-posterior and dorsal-ventral patterning during mDA neuron development in vitro (Tremolanti et al, manuscript under revision)
2) Develop a cell-replacement strategy for PD based on the transplantation of hPSC-derived mDA progenitors differentiating into A9/SNc neurons. In an in silico forward programming simulation we used data from the literature and identified a number of candidates which were also hand-curated in the targeted transcription factor sgRNA library that was used for the loss of function screen in aim 1.
Transplantation of human PSC derived mDA progenitors into hemiparkinsonian immunocompromised mice, which previously have not been utilized in intracranial grafting experiments, restored motor function. Progenitors were shown to mature into a large percentage of DA neurons expressing canonical markers for A9-subtype mDA neurons, which was further confirmed by a comparison of the transcriptional profiles to those of native adult human mDA cells showing a successful generation of in vivo differentiated cells resembling human SOX6 mDA neurons (Garcia Swinburn et al, submitted manuscript).
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. We improved a previous protocol which resulted in a 70% increase of TH + neurons. We also performed single cell RNA sequencing to compare the in vivo reprogrammed mDA neurons to human embryonic mDA neurons. This analysis is under way.
Our work resulted in a total of 6 publications, 1 manuscript in press, 1 manuscript under revision, 1 submitted manuscript, 2 manuscripts in preparation.
Our work has uncovered the molecular diversity of dopaminergic neurons in the adult human midbrain which was a major gap. This work provides ground-breaking insight into the molecular mechanisms that control the identity and maintenance of human A9/SNc dopaminergic neurons. We developed a protocol for improved generation of dopaminergic neurons from astrocytes and improved a protocol for cell replacement strategies in a rodent model of PD. This project contributed to finding new precision cell-replacement therapies for PD leading to a paradigm shift to only replace the specific cell type/s required to restore the functions lost to PD.