Abstract:Translating new cell-based therapies to the clinic for patients with neurodegenerative disorders is complex. It involves pre-clinical testing of the cellular product and discussions with several regulatory agencies, as well as ethical debates. In an attempt to support efforts around the world, we set up a global consortium that brings together the major funded teams working on developing a stem cell-derived neural transplantation therapy for Parkinson’s disease (PD). This consortium, G-Force PD, involves teams… Show more
“…CP cells directed to mesendoderm fates were also indistinguishable in the extent and kinetics of differentiation (Figures 3D–3F). Both of these cell fates are relatively immature and therefore easier to make, so we attempted to make midbrain dopamine neurons from CP cells, a cell type that our group manufactured in clinically compatible conditions as part of Lorenz Studer's NYSTEM consortium group (Barker et al., 2015). WA09 CP cells efficiently created FOXA2/TH double-positive, post-mitotic midbrain dopamine neurons from a clinically compatible “standard operating procedure” and display a gene expression profile similar to that of neurons derived from control cells (Figures 3I and 3J).Figure 3The Kinetics and Extent of Directed Differentiation of CryoPaused Cells(A) OCT4 and PAX6 flow-cytometry quantification during neural induction (n = 5; values for independent biological replicates shown as mean ± SD).(B and C) Representative flow cytometry (B) and immunofluorescence (C) of OCT4 and PAX6 expression at 0, 3, 6, and 9 days after neural induction.(D) OCT4 and Brachyury expression quantified by flow cytometry during mesendodermal induction (n = 3; values for independent biological replicates shown as mean ± SD).(E and F) Representative flow cytometry (E) and immunofluorescence (F) at 0, 2, and 4 days after mesendoderm induction.(G) Viability of CryoPaused cells post thaw when frozen at 1, 5, 10, 20, and 30 million cells/mL.…”
SummaryHuman pluripotent stem cells (PSCs) provide an unlimited cell source for cell therapies and disease modeling. Despite their enormous power, technical aspects have hampered reproducibility. Here, we describe a modification of PSC workflows that eliminates a major variable for nearly all PSC experiments: the quality and quantity of the PSC starting material. Most labs continually passage PSCs and use small quantities after expansion, but the “just-in-time” nature of these experiments means that quality control rarely happens before use. Lack of quality control could compromise PSC quality, sterility, and genetic integrity, which creates a variable that might affect results. This method, called CryoPause, banks PSCs as single-use, cryopreserved vials that can be thawed and immediately used in experiments. Each CryoPause bank provides a consistent source of PSCs that can be pre-validated before use to reduce the possibility that high levels of spontaneous differentiation, contamination, or genetic integrity will compromise an experiment.
“…CP cells directed to mesendoderm fates were also indistinguishable in the extent and kinetics of differentiation (Figures 3D–3F). Both of these cell fates are relatively immature and therefore easier to make, so we attempted to make midbrain dopamine neurons from CP cells, a cell type that our group manufactured in clinically compatible conditions as part of Lorenz Studer's NYSTEM consortium group (Barker et al., 2015). WA09 CP cells efficiently created FOXA2/TH double-positive, post-mitotic midbrain dopamine neurons from a clinically compatible “standard operating procedure” and display a gene expression profile similar to that of neurons derived from control cells (Figures 3I and 3J).Figure 3The Kinetics and Extent of Directed Differentiation of CryoPaused Cells(A) OCT4 and PAX6 flow-cytometry quantification during neural induction (n = 5; values for independent biological replicates shown as mean ± SD).(B and C) Representative flow cytometry (B) and immunofluorescence (C) of OCT4 and PAX6 expression at 0, 3, 6, and 9 days after neural induction.(D) OCT4 and Brachyury expression quantified by flow cytometry during mesendodermal induction (n = 3; values for independent biological replicates shown as mean ± SD).(E and F) Representative flow cytometry (E) and immunofluorescence (F) at 0, 2, and 4 days after mesendoderm induction.(G) Viability of CryoPaused cells post thaw when frozen at 1, 5, 10, 20, and 30 million cells/mL.…”
SummaryHuman pluripotent stem cells (PSCs) provide an unlimited cell source for cell therapies and disease modeling. Despite their enormous power, technical aspects have hampered reproducibility. Here, we describe a modification of PSC workflows that eliminates a major variable for nearly all PSC experiments: the quality and quantity of the PSC starting material. Most labs continually passage PSCs and use small quantities after expansion, but the “just-in-time” nature of these experiments means that quality control rarely happens before use. Lack of quality control could compromise PSC quality, sterility, and genetic integrity, which creates a variable that might affect results. This method, called CryoPause, banks PSCs as single-use, cryopreserved vials that can be thawed and immediately used in experiments. Each CryoPause bank provides a consistent source of PSCs that can be pre-validated before use to reduce the possibility that high levels of spontaneous differentiation, contamination, or genetic integrity will compromise an experiment.
“…More recently, similar principles have been implemented to differentiate human‐induced pluripotent stem cells to mDA neurons (Doi et al, ) and to engineer mDA cells whose activity can be regulated either with optogenetic tools (Steinbeck et al, ) or with designer receptors exclusively activated by designer drugs (Chen et al, ). Current efforts by several groups and the G‐FORCE Consortia (Barker et al , ) now focus on the development of clinical grade hPSCs cells for CRT in PD.…”
Wnt signalling is a highly conserved pathway across species that is critical for normal development and is deregulated in multiple disorders including cancer and neurodegenerative diseases. Wnt signalling is critically required for midbrain dopaminergic (mDA) neuron development and maintenance. Understanding the molecular processes controlled by Wnt signalling may thus hold the key to understand the physiopathology and to develop novel therapies aimed at preventing the loss of mDA neurons in Parkinson's disease (PD). Pharmacological tools to activate Wnt signalling have been used to translate in vivo developmental processes into protocols for the generation of bona fide mDA neurons from human pluripotent stem cells. Moreover, these protocols are currently being fine-tuned to generate mDA neurons for clinical trials in PD. At the same time, a vast amount of molecular details of Wnt signalling continues to emerge and remains to be implemented into new protocols. We hereby review novel pharmacological tools to activate Wnt signalling and how single-cell RNA-sequencing is contributing to unravel the complexity of this pathway in the developing human ventral midbrain, generating novel hypotheses and identifying new players and opportunities to further improve cell replacement therapy for PD.
“…We expect that as techniques improve in neural differentiation, culture purity and experimental control will only improve. The most comprehensive examples of postmitotic neuron purity can be found in the literature from midbrain dopaminergic neurons of the A9 type, resembling those dopamine‐producing cells from the substantia nigra, since significant resources have been deployed to attempt to make transplantable cells to treat Parkinson's disease . What is reassuring from this literature is not that cell purity has reached 100% for dopamine‐producing cells, but that the technology development that has been fostered by active investigations suggest a clear trajectory toward improved cell purity.…”
Section: Identification and Selection Of Neural Populationsmentioning
Stem and derivative cells induced from somatic tissues are a critical tool for disease modeling but significant technical hurdles hamper their use. The purpose of this review is to provide an overview of pitfalls and mitigation strategies for the nonstem cell biologist using induced pluripotent stem cells and investigating neurodevelopmental disorders. What sample sizes are reasonable? What derivation and purification protocols should be used to make human neurons? In what way should gene editing technologies be used to support discoveries? What kinds of preclinical studies are the most feasible? It is hoped that this roadmap will provide the necessary details for experimental planning and execution for those less familiar in the area of stem cell disease modeling. High-quality human preclinical models will allow for the discovery of molecular and cellular phenotypes specific to different neurodevelopmental disorders, and may provide the assays to advance translational medicine for unmet medical needs. K E Y W O R D S genetic engineering, neurodevelopmental disorders, neuronal induction, somatic cell reprogramming, stem cells, therapeutics
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