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Differentiation from human pluripotent stem cells of cortical neurons of the superficial layers amenable to psychiatric disease modeling and high-throughput drug screening.

Boissart C, Poulet A, Georges P, Darville H, Julita E, Delorme R, Bourgeron T, Peschanski M, Benchoua A - Transl Psychiatry (2013)

Bottom Line: Addressing this issue, we have successfully captured a stable population of self-renewing late cortical progenitors (LCPs) that synchronously and massively differentiate into glutamatergic cortical neurons of the upper layers.This has allowed us to analyze the capability of LCPs at differentiating into post mitotic neurons as well as extending and branching neurites in response to a collection of selected bioactive molecules.LCPs and cortical neurons of the upper layers were successfully produced from patient-derived-induced PSC, indicating that this system enables functional studies of individual-specific cortical neurons ex vivo for disease modeling and therapeutic purposes.

View Article: PubMed Central - PubMed

Affiliation: Neuroplasticity and Therapeutics, CECS, I-STEM, AFM, Evry Cedex, France.

ABSTRACT
Cortical neurons of the superficial layers (II-IV) represent a pivotal neuronal population involved in the higher cognitive functions of the human and are particularly affected by psychiatric diseases with developmental manifestations such as schizophrenia and autism. Differentiation protocols of human pluripotent stem cells (PSC) into cortical neurons have been achieved, opening the way to in vitro modeling of neuropsychiatric diseases. However, these protocols commonly result in the asynchronous production of neurons typical for the different layers of the cortex within an extended period of culture, thus precluding the analysis of specific subtypes of neurons in a standardized manner. Addressing this issue, we have successfully captured a stable population of self-renewing late cortical progenitors (LCPs) that synchronously and massively differentiate into glutamatergic cortical neurons of the upper layers. The short time course of differentiation into neurons of these progenitors has made them amenable to high-throughput assays. This has allowed us to analyze the capability of LCPs at differentiating into post mitotic neurons as well as extending and branching neurites in response to a collection of selected bioactive molecules. LCPs and cortical neurons of the upper layers were successfully produced from patient-derived-induced PSC, indicating that this system enables functional studies of individual-specific cortical neurons ex vivo for disease modeling and therapeutic purposes.

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Related in: MedlinePlus

Overview of the timelines of the differentiation protocol. Phase I (days 0–35) corresponds to the steps of differentiation of human pluripotent stem cells (hPSC) into early cortical progenitors, the subsequent phenotypic transition to late cortical progenitors (LCP) and further amplification to create a large cell bank. After this phase, neuronal differentiation into cortical projection neurons is achieved in 14–17 days (phase II). As neuronal differentiation can be started directly from the stock of frozen LCP, the phase 1 does not need to be completed at each experiment. Liquid N2; liquid nitrogen.
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fig6: Overview of the timelines of the differentiation protocol. Phase I (days 0–35) corresponds to the steps of differentiation of human pluripotent stem cells (hPSC) into early cortical progenitors, the subsequent phenotypic transition to late cortical progenitors (LCP) and further amplification to create a large cell bank. After this phase, neuronal differentiation into cortical projection neurons is achieved in 14–17 days (phase II). As neuronal differentiation can be started directly from the stock of frozen LCP, the phase 1 does not need to be completed at each experiment. Liquid N2; liquid nitrogen.

Mentions: Superficial layer neurons represent a pivotal neuronal population particularly affected by psychiatric diseases.4, 5, 6, 26, 27 As expanded superficial layers of the cortex are among the most distinguishing features of the human brain, modeling the impact of psychiatric diseases using these neurons may help revealing human-specific aspects. Superficial layers of the cortex are poorly developed in rodents as compared with humans, a situation replicated in vitro when corticogenesis is recapitulated from PSCs.11, 12 Superficial layers neurons are late-born cells and, accordingly, their derivation from human PSCs only occurs after an extended period of differentiation. In their pioneer study, Shi et al.12, 28 described a protocol recapitulating key features of corticogenesis starting from human PSC. In this three-stage process, PSC first differentiated to cortical progenitors cells resembling the cortical sub-ventricular radial glia cells that will spontaneously produce intermediate progenitors and the different types of projection neurons of the deep and superficial layers in stereotypical temporal order reminiscent of in vivo corticogenesis. Accordingly, superficial layer neurons appeared in small amount (∼ 30%) after 80–90 days of differentiation, mixed with undifferentiated progenitors and earlier-born neurons of the deeper layers. Although very elegant, this approach, which was to our knowledge the only producing superficial layer neurons from hPSC, precluded a robust and a systematic study of the impact of a disease state on genesis and maturation of these neurons because of this protracted time of differentiation. Consequently, studies of neurons derived from neuropsychiatric patients iPSC were so far conducted using quicker protocols of differentiation leading to a mixture of GABAergic and glutamatergic neurons with not known regional identity.29, 30 This temporal challenge is also particularly critical when addressing drug discovery using HTS as this powerful technique requires that the terminal differentiation and maturation steps of these neurons occurs in a rapid, reproducible and synchronous manner. The approach described here allowed us to differentiate and stabilize cortical precursors that could be largely amplified and preferentially produced glutamatergic neurons of the superficial layers. This was achieved by mimicking the chronobiology of the human corticogenesis, that is, by preventing the spontaneous neuronal differentiation of early NEPs patterned to adopt a dorsal telencephalic fate while promoting active cell divisions. Accordingly, the neurogenic potential of the PSC-derived cortical precursors changed from deep to superficial layers after the period of active division. Consequently, large amounts (>70%) of superficial layers neurons can be produced in less than 50 days starting from PSC (instead of 80–90 days). Importantly, as the self-renewing LCP can be cultivated and amplified as single-cell culture, they can be stored frozen as very large banks. Terminal differentiation can therefore be restarted directly from the LCP at any time, and superficial layer neurons were produced in less than 17 days (Figure 6).


Differentiation from human pluripotent stem cells of cortical neurons of the superficial layers amenable to psychiatric disease modeling and high-throughput drug screening.

Boissart C, Poulet A, Georges P, Darville H, Julita E, Delorme R, Bourgeron T, Peschanski M, Benchoua A - Transl Psychiatry (2013)

Overview of the timelines of the differentiation protocol. Phase I (days 0–35) corresponds to the steps of differentiation of human pluripotent stem cells (hPSC) into early cortical progenitors, the subsequent phenotypic transition to late cortical progenitors (LCP) and further amplification to create a large cell bank. After this phase, neuronal differentiation into cortical projection neurons is achieved in 14–17 days (phase II). As neuronal differentiation can be started directly from the stock of frozen LCP, the phase 1 does not need to be completed at each experiment. Liquid N2; liquid nitrogen.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3756296&req=5

fig6: Overview of the timelines of the differentiation protocol. Phase I (days 0–35) corresponds to the steps of differentiation of human pluripotent stem cells (hPSC) into early cortical progenitors, the subsequent phenotypic transition to late cortical progenitors (LCP) and further amplification to create a large cell bank. After this phase, neuronal differentiation into cortical projection neurons is achieved in 14–17 days (phase II). As neuronal differentiation can be started directly from the stock of frozen LCP, the phase 1 does not need to be completed at each experiment. Liquid N2; liquid nitrogen.
Mentions: Superficial layer neurons represent a pivotal neuronal population particularly affected by psychiatric diseases.4, 5, 6, 26, 27 As expanded superficial layers of the cortex are among the most distinguishing features of the human brain, modeling the impact of psychiatric diseases using these neurons may help revealing human-specific aspects. Superficial layers of the cortex are poorly developed in rodents as compared with humans, a situation replicated in vitro when corticogenesis is recapitulated from PSCs.11, 12 Superficial layers neurons are late-born cells and, accordingly, their derivation from human PSCs only occurs after an extended period of differentiation. In their pioneer study, Shi et al.12, 28 described a protocol recapitulating key features of corticogenesis starting from human PSC. In this three-stage process, PSC first differentiated to cortical progenitors cells resembling the cortical sub-ventricular radial glia cells that will spontaneously produce intermediate progenitors and the different types of projection neurons of the deep and superficial layers in stereotypical temporal order reminiscent of in vivo corticogenesis. Accordingly, superficial layer neurons appeared in small amount (∼ 30%) after 80–90 days of differentiation, mixed with undifferentiated progenitors and earlier-born neurons of the deeper layers. Although very elegant, this approach, which was to our knowledge the only producing superficial layer neurons from hPSC, precluded a robust and a systematic study of the impact of a disease state on genesis and maturation of these neurons because of this protracted time of differentiation. Consequently, studies of neurons derived from neuropsychiatric patients iPSC were so far conducted using quicker protocols of differentiation leading to a mixture of GABAergic and glutamatergic neurons with not known regional identity.29, 30 This temporal challenge is also particularly critical when addressing drug discovery using HTS as this powerful technique requires that the terminal differentiation and maturation steps of these neurons occurs in a rapid, reproducible and synchronous manner. The approach described here allowed us to differentiate and stabilize cortical precursors that could be largely amplified and preferentially produced glutamatergic neurons of the superficial layers. This was achieved by mimicking the chronobiology of the human corticogenesis, that is, by preventing the spontaneous neuronal differentiation of early NEPs patterned to adopt a dorsal telencephalic fate while promoting active cell divisions. Accordingly, the neurogenic potential of the PSC-derived cortical precursors changed from deep to superficial layers after the period of active division. Consequently, large amounts (>70%) of superficial layers neurons can be produced in less than 50 days starting from PSC (instead of 80–90 days). Importantly, as the self-renewing LCP can be cultivated and amplified as single-cell culture, they can be stored frozen as very large banks. Terminal differentiation can therefore be restarted directly from the LCP at any time, and superficial layer neurons were produced in less than 17 days (Figure 6).

Bottom Line: Addressing this issue, we have successfully captured a stable population of self-renewing late cortical progenitors (LCPs) that synchronously and massively differentiate into glutamatergic cortical neurons of the upper layers.This has allowed us to analyze the capability of LCPs at differentiating into post mitotic neurons as well as extending and branching neurites in response to a collection of selected bioactive molecules.LCPs and cortical neurons of the upper layers were successfully produced from patient-derived-induced PSC, indicating that this system enables functional studies of individual-specific cortical neurons ex vivo for disease modeling and therapeutic purposes.

View Article: PubMed Central - PubMed

Affiliation: Neuroplasticity and Therapeutics, CECS, I-STEM, AFM, Evry Cedex, France.

ABSTRACT
Cortical neurons of the superficial layers (II-IV) represent a pivotal neuronal population involved in the higher cognitive functions of the human and are particularly affected by psychiatric diseases with developmental manifestations such as schizophrenia and autism. Differentiation protocols of human pluripotent stem cells (PSC) into cortical neurons have been achieved, opening the way to in vitro modeling of neuropsychiatric diseases. However, these protocols commonly result in the asynchronous production of neurons typical for the different layers of the cortex within an extended period of culture, thus precluding the analysis of specific subtypes of neurons in a standardized manner. Addressing this issue, we have successfully captured a stable population of self-renewing late cortical progenitors (LCPs) that synchronously and massively differentiate into glutamatergic cortical neurons of the upper layers. The short time course of differentiation into neurons of these progenitors has made them amenable to high-throughput assays. This has allowed us to analyze the capability of LCPs at differentiating into post mitotic neurons as well as extending and branching neurites in response to a collection of selected bioactive molecules. LCPs and cortical neurons of the upper layers were successfully produced from patient-derived-induced PSC, indicating that this system enables functional studies of individual-specific cortical neurons ex vivo for disease modeling and therapeutic purposes.

Show MeSH
Related in: MedlinePlus