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Editor | Qi Parkinson's disease (Parkinson's disease, PD) is one of the most common neurodegenerative diseases worldwide.
Its main characteristics include motor symptoms, such as bradykinesia, stiffness, resting tremor, and postural instability.
Accompanied by the appearance of some non-motor symptoms.
Current drug treatment is mainly through dopamine supplements or agonists, or by inhibiting the degradation of dopamine to increase the level of dopamine in the brain.
However, although this treatment strategy is effective, it is not long-lasting and lacks neuroprotection.
The use of fetal tissue sources for dopamine neuron transplantation has achieved long-term benefits in some patients, but the application of this method is limited by ethical issues and the robustness of the efficacy.
Therefore, there is an urgent need to develop an alternative therapy that is easy to obtain and expand.
Human pluripotent stem cells (hPSCs) have become the most promising cell source for midbrain dopamine (mDA) neurons in PD patients.
In PD models of mice, rats, and monkeys, the robust implantation of in vitro hPSC-derived mDA neurons requires the development of a floor-derived midbrain dopamine neuron differentiation protocol, and all of these protocols are based on WNT signal activators (usually GSK3).
The combined effect of the inhibitor CHIR99021) and the strong activation of SHH signals to trigger the induction of the midbrain floor plate and the neurogenic transformation into mDA neurons.
It should be noted that there are considerable differences in the time and concentration of using CHIR99021 and the additional inducer of mDA neuron properties-fibroblast growth factor 8 (FGF8) between the various protocols.
So how to avoid the pollution of other types of neurons in the process of inducing differentiation, and how to develop a robust and scalable mDA neuron induction program for clinical treatment is particularly important.
Recently, the Lorenz Studer team from the Memorial Sloan Kettering Cancer Center in the United States and the Viviane Tabar team (Tae Wan Kim and Jinghua Piao are co-authored) published a titled "Biphasic Activation of WNT Signaling Facilitates the Derivation" in Cell Stem Cell.
"of Midbrain Dopamine Neurons from hESCs for Translational Use", proposes a biphasic WNT signal activation to optimize midbrain dopamine neuron differentiation, which increases the expression of midbrain markers (such as EN1) while also minimizing In addition to the expression of other neuron and non-neuronal lineage markers, the resulting neurons exhibit a high degree of mDA neuron molecular, biochemical and electrophysiological properties.
More importantly, cryopreserved mDA precursor neurons can be successfully transplanted into the 6-OHDA-induced PD rat model, and restore the damaged side rotation behavior induced by amphetamine.
This program is the basis for clinical-level mDA neuron generation and safety and effectiveness research.
Previous studies have widely used CHIR99021 to induce mDA neurons.
One of the problems is that at different stages of differentiation, even with the same concentration of CHIR99021, the induction of midbrain markers such as EN1 may change greatly.
This batch-to-batch difference, as well as the sensitivity to small changes in the concentration of CHIR99021, makes the process of inducing mDA frequently contaminated by the differentiation and fate of other neuron types such as the hindbrain or diencephalon.
Although FGF8b is believed to be able to reliably induce the expression of midbrain markers, it also seems to be related to the time of use.
That is to say, exposure to FGF8b in the early stage of mDA pattern formation does not significantly increase the expression of midbrain markers.
In the later stage, EN1 levels can be increased, and similarly, it can also induce cell type contamination of non-mDA neurons.
In addition, WNT signals also have time- and dose-dependent effects in different developmental stages of the midbrain to drive neurons to different fates.
Based on the above findings, the researchers used transcriptome analysis and CHIP-seq to verify the biphasic activation of WNT signals during the neural differentiation process of hPSCs (that is, early low Chir concentration triggers the induction of midbrain/hindbrain characteristics → high Chir concentration (Chir-boost) targeting the midbrain rather than the induction of the hindbrain characteristics) the effectiveness of specific induction of mDA neurons and the inhibitory effect on non-mDA markers.
Of course, in addition to confirming the markers, the researchers also conducted further functional studies on the induced mDA neurons.
The patch clamp experiment showed that its electrophysiological characteristics showed similarities to those of rodent midbrain DA neurons.
HPLC analysis also showed that neurons on the 60th day were able to synthesize and release DA neurotransmitter in a stimulated and Ca2+ dependent manner.
Next is the most important issue of clinical transformation.
Taking into account the timeliness and possible batch-to-batch differences, mDA neurons cannot always be "made-in-place", and it is best to use cryopreserved ready-made ones.
Products in order to conduct extensive and repeated product testing before clinical use.
In addition, the in vivo potential of mDA neuron precursors needs to be considered, and its distribution, toxicity, tumorigenicity and functionality need to be evaluated.
Therefore, these teams published another article titled "Preclinical Efficacy and Safety of a Human Embryonic Stem Cell-Derived Midbrain Dopamine Progenitor Product, MSK-DA01" in the same period of Cell Stem Cell.
In this article, I elaborated on the preservation and reuse efficiency of large-scale cryopreserved clinical-grade mDA neuron precursor cells (named MSK-DA01), and explained the design and results of in vivo studies in rodents.
In this article, the researchers first focused on the production protocol and quality control issues of MSK-DA01 cells, specifically, including cell morphology, proliferation and cell viability analysis, as well as any undifferentiated products derived from PSCs The pollution problem of PSCs.
Subsequently, the researchers tested the in vivo survival rate and mDA differentiation of four batches of MSK-DA01, and injected the cells unilaterally into the striatum of severe combined immunodeficiency/non-obese diabetic NSG mice through a variety of human cell markers Identify the formation of the graft at the injection site.
Next, the researchers focused on a long-term efficacy verification, the purpose is to analyze the effect of MSK-DA01 on the animal behavior of 6-OHDA-induced nude mice (NIH-Foxn1rnu), as well as the survival, differentiation and adverse effects of the graft.
Wait for the situation.
The results of the study show that the injection can significantly improve the rotation behavior of the injured side of the mice.
Pathological examination after the euthanasia of the mice shows that there are a large number of well-differentiated human dopamine neurons, and good connections are established with the surrounding tissues.
In addition, the distribution test showed that the transplanted MSK-DA01 cells were confined to the brain.
Except for small glial hyperplasia and traumatic changes at the injection site, no vascular invasion or other pathologically meaningful lesions were found, and no MSK-DA01 transplantation was found.
The tumorigenicity and so on.
In general, these two articles propose a method of deriving mDA neurons from hPSCs and using an optimized scheme suitable for mass production, preservation and transplantation.
This is a project completed in nearly ten years of hard work.
works.
The data in the article on the biodistribution, toxicity, tumorigenicity, and efficacy outcome of the graft support the initiation of clinical trials.
In addition, the author mentioned that they have established a team including neurologists, neurosurgeons, ethicists, etc.
, to formulate clinical trial drafts, and they have also received funding support.
Of course, until the actual clinical application, there will be a series of additional challenges, but these difficulties will be worthwhile in the face of the ultimate conversion value.
Original link https://doi.
org/10.
1016/j.
stem.
2021.
01.
005https://doi.
org/10.
1016/j.
stem.
2021.
01.
004 Plate maker: Qi Jiang
Its main characteristics include motor symptoms, such as bradykinesia, stiffness, resting tremor, and postural instability.
Accompanied by the appearance of some non-motor symptoms.
Current drug treatment is mainly through dopamine supplements or agonists, or by inhibiting the degradation of dopamine to increase the level of dopamine in the brain.
However, although this treatment strategy is effective, it is not long-lasting and lacks neuroprotection.
The use of fetal tissue sources for dopamine neuron transplantation has achieved long-term benefits in some patients, but the application of this method is limited by ethical issues and the robustness of the efficacy.
Therefore, there is an urgent need to develop an alternative therapy that is easy to obtain and expand.
Human pluripotent stem cells (hPSCs) have become the most promising cell source for midbrain dopamine (mDA) neurons in PD patients.
In PD models of mice, rats, and monkeys, the robust implantation of in vitro hPSC-derived mDA neurons requires the development of a floor-derived midbrain dopamine neuron differentiation protocol, and all of these protocols are based on WNT signal activators (usually GSK3).
The combined effect of the inhibitor CHIR99021) and the strong activation of SHH signals to trigger the induction of the midbrain floor plate and the neurogenic transformation into mDA neurons.
It should be noted that there are considerable differences in the time and concentration of using CHIR99021 and the additional inducer of mDA neuron properties-fibroblast growth factor 8 (FGF8) between the various protocols.
So how to avoid the pollution of other types of neurons in the process of inducing differentiation, and how to develop a robust and scalable mDA neuron induction program for clinical treatment is particularly important.
Recently, the Lorenz Studer team from the Memorial Sloan Kettering Cancer Center in the United States and the Viviane Tabar team (Tae Wan Kim and Jinghua Piao are co-authored) published a titled "Biphasic Activation of WNT Signaling Facilitates the Derivation" in Cell Stem Cell.
"of Midbrain Dopamine Neurons from hESCs for Translational Use", proposes a biphasic WNT signal activation to optimize midbrain dopamine neuron differentiation, which increases the expression of midbrain markers (such as EN1) while also minimizing In addition to the expression of other neuron and non-neuronal lineage markers, the resulting neurons exhibit a high degree of mDA neuron molecular, biochemical and electrophysiological properties.
More importantly, cryopreserved mDA precursor neurons can be successfully transplanted into the 6-OHDA-induced PD rat model, and restore the damaged side rotation behavior induced by amphetamine.
This program is the basis for clinical-level mDA neuron generation and safety and effectiveness research.
Previous studies have widely used CHIR99021 to induce mDA neurons.
One of the problems is that at different stages of differentiation, even with the same concentration of CHIR99021, the induction of midbrain markers such as EN1 may change greatly.
This batch-to-batch difference, as well as the sensitivity to small changes in the concentration of CHIR99021, makes the process of inducing mDA frequently contaminated by the differentiation and fate of other neuron types such as the hindbrain or diencephalon.
Although FGF8b is believed to be able to reliably induce the expression of midbrain markers, it also seems to be related to the time of use.
That is to say, exposure to FGF8b in the early stage of mDA pattern formation does not significantly increase the expression of midbrain markers.
In the later stage, EN1 levels can be increased, and similarly, it can also induce cell type contamination of non-mDA neurons.
In addition, WNT signals also have time- and dose-dependent effects in different developmental stages of the midbrain to drive neurons to different fates.
Based on the above findings, the researchers used transcriptome analysis and CHIP-seq to verify the biphasic activation of WNT signals during the neural differentiation process of hPSCs (that is, early low Chir concentration triggers the induction of midbrain/hindbrain characteristics → high Chir concentration (Chir-boost) targeting the midbrain rather than the induction of the hindbrain characteristics) the effectiveness of specific induction of mDA neurons and the inhibitory effect on non-mDA markers.
Of course, in addition to confirming the markers, the researchers also conducted further functional studies on the induced mDA neurons.
The patch clamp experiment showed that its electrophysiological characteristics showed similarities to those of rodent midbrain DA neurons.
HPLC analysis also showed that neurons on the 60th day were able to synthesize and release DA neurotransmitter in a stimulated and Ca2+ dependent manner.
Next is the most important issue of clinical transformation.
Taking into account the timeliness and possible batch-to-batch differences, mDA neurons cannot always be "made-in-place", and it is best to use cryopreserved ready-made ones.
Products in order to conduct extensive and repeated product testing before clinical use.
In addition, the in vivo potential of mDA neuron precursors needs to be considered, and its distribution, toxicity, tumorigenicity and functionality need to be evaluated.
Therefore, these teams published another article titled "Preclinical Efficacy and Safety of a Human Embryonic Stem Cell-Derived Midbrain Dopamine Progenitor Product, MSK-DA01" in the same period of Cell Stem Cell.
In this article, I elaborated on the preservation and reuse efficiency of large-scale cryopreserved clinical-grade mDA neuron precursor cells (named MSK-DA01), and explained the design and results of in vivo studies in rodents.
In this article, the researchers first focused on the production protocol and quality control issues of MSK-DA01 cells, specifically, including cell morphology, proliferation and cell viability analysis, as well as any undifferentiated products derived from PSCs The pollution problem of PSCs.
Subsequently, the researchers tested the in vivo survival rate and mDA differentiation of four batches of MSK-DA01, and injected the cells unilaterally into the striatum of severe combined immunodeficiency/non-obese diabetic NSG mice through a variety of human cell markers Identify the formation of the graft at the injection site.
Next, the researchers focused on a long-term efficacy verification, the purpose is to analyze the effect of MSK-DA01 on the animal behavior of 6-OHDA-induced nude mice (NIH-Foxn1rnu), as well as the survival, differentiation and adverse effects of the graft.
Wait for the situation.
The results of the study show that the injection can significantly improve the rotation behavior of the injured side of the mice.
Pathological examination after the euthanasia of the mice shows that there are a large number of well-differentiated human dopamine neurons, and good connections are established with the surrounding tissues.
In addition, the distribution test showed that the transplanted MSK-DA01 cells were confined to the brain.
Except for small glial hyperplasia and traumatic changes at the injection site, no vascular invasion or other pathologically meaningful lesions were found, and no MSK-DA01 transplantation was found.
The tumorigenicity and so on.
In general, these two articles propose a method of deriving mDA neurons from hPSCs and using an optimized scheme suitable for mass production, preservation and transplantation.
This is a project completed in nearly ten years of hard work.
works.
The data in the article on the biodistribution, toxicity, tumorigenicity, and efficacy outcome of the graft support the initiation of clinical trials.
In addition, the author mentioned that they have established a team including neurologists, neurosurgeons, ethicists, etc.
, to formulate clinical trial drafts, and they have also received funding support.
Of course, until the actual clinical application, there will be a series of additional challenges, but these difficulties will be worthwhile in the face of the ultimate conversion value.
Original link https://doi.
org/10.
1016/j.
stem.
2021.
01.
005https://doi.
org/10.
1016/j.
stem.
2021.
01.
004 Plate maker: Qi Jiang
