Development and Characterization of Human iPSC-Derived Forebrain Neuron Precursor Cells
What are Forebrain Neurons?
Human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), possess the ability to differentiate into various cell types derived from all three embryonic germ layers. Among these differentiated cell types, forebrain neurons are essential for understanding the mechanisms of neuronal differentiation and the development of neurodegenerative diseases. These neurons offer valuable insights into the functioning of the forebrain, a critical region responsible for cognitive functions, sensory processing, and motor control.
Forebrain neurons are specialized cells that emerge during brain development and continue to play vital roles throughout life. They originate from neural precursors that proliferate and differentiate to form the diverse array of neurons in the forebrain. These neurons are identified by the expression of specific molecular markers such as FOXG1 and MAP2, which signify their forebrain identity and advanced developmental stage.
Forebrain neurons can be classified into two primary types: glutamatergic (excitatory) and GABAergic (inhibitory) neurons. Glutamatergic neurons release the neurotransmitter glutamate and are pivotal in excitatory signaling within the brain, crucial for learning, memory, and overall brain function. GABAergic neurons, conversely, release gamma-aminobutyric acid (GABA) and are essential for inhibitory signaling, helping to regulate neuronal excitability and maintain the balance between excitation and inhibition in neural circuits.
In the adult brain, neural precursors persist in regions such as the subventricular zone (SVZ) and the hippocampus, both of which are crucial for the generation of forebrain neurons. The SVZ lines the lateral ventricles and serves as a source of new neurons, while the hippocampus is integral to learning and memory. Throughout life, these regions contribute to the brain's plasticity by generating new forebrain neurons in response to injury or disease, thereby supporting brain repair and functional recovery.
Forebrain neurons are of significant interest in clinical research due to their relevance in a variety of neurological conditions. They are crucial for studying diseases such as Alzheimer's, epilepsy, and various forms of neurodevelopmental disorders, where the normal function of the forebrain is disrupted. Clinical trials and research studies utilize forebrain neurons to explore therapeutic approaches, including drug screening, neurotoxicity testing, and regenerative medicine.
Recent advancements in neural differentiation techniques, particularly the dual-SMAD inhibition method, have significantly enhanced the efficiency of generating forebrain neurons from hPSCs. This method employs inhibitors like SB431542 and Noggin to block SMAD signaling pathways, enabling rapid and reliable neural induction under defined conditions (Chambers et al., 2009). In addition, protocols for inducing specific neuron types, such as the GABAergic neurons and excitatory glutamatergic neurons, have further refined the process. Sun et al. (2016) identified factors like ASCL1, DLX2, and LHX6, along with microRNAs miR-9/9* and miR-124, to robustly induce hPSCs into GABAergic neurons with mature electrophysiological properties. Similarly, Ho et al. (2015) described a method using Neurogenin 2 (Ngn2) to rapidly induce iPSC-derived neural progenitor cells into functional excitatory neurons within two weeks, yielding over 90% MAP2AB-positive neurons expressing glutamatergic synaptic proteins. These advancements have streamlined the generation of diverse forebrain neurons, making it easier for researchers to obtain high-quality cells for their studies.
One exciting development in the field of forebrain neuron research is a Phase 1/2 clinical trial currently underway, utilizing hPSC-derived GABA-secreting interneurons to treat drug-resistant unilateral mesial temporal lobe epilepsy. Led by Neurona Therapeutics in the USA, this study (NCT05135091) explores the potential of these neurons to provide relief for patients who have not responded to conventional therapies. The trial involves an approach where patients receive a single intracerebral administration of NRTX-1001, a neural cell therapy product derived from human ESCs. These neurons, engineered to secrete the inhibitory neurotransmitter GABA, aim to restore balance in the brain’s electrical activity, potentially reducing seizure frequency and severity. Participants are closely monitored for safety and efficacy, with the hope that this advanced treatment could pave the way for new therapeutic strategies in combating severe forms of epilepsy.
In summary, forebrain neurons are a fundamental cell type for neuroscience research, offering profound insights into brain function and disease. Their expanding role in clinical research highlights their potential in developing novel therapeutic strategies for neurological disorders.
Human iPSC-Derived Forebrain Neurons from STEMCELL Technologies
STEMCELL Technologies has developed cryopreserved Human iPSC-Derived Forebrain Neuron Precursor Cells (Catalog no. 200-0770) for academic and commercial research purposes. Human iPSC-Derived Forebrain Neuron Precursor Cells were manufactured from the Healthy Control Human iPSC Line, Female, SCTi003-A (Catalog #200-0511), using the STEMdiff? SMADi Neural Induction Kit (Catalog #08581) and STEMdiff? Forebrain Neuron Differentiation Kit (Catalog #08600). The resulting cells express high levels of neuronal markers MAP2 and GABA, the forebrain neuron marker FOXG1, and pre-synaptic marker Synapsin.
The Product Information Sheet (PIS) for Human iPSC-Derived Forebrain Neuron Precursor Cells can be found here.
Advantages:
Cell Line Information - SCTi003-A
Forebrain neuron precursor cells were differentiated from the Healthy Control Human iPSC Line, SCTi003-A (Catalog no. 200-0511); a cell line derived from peripheral blood mononuclear cells (PBMCs) from a 48-year-old female donor. Extensive quality control procedures are undertaken in our iPSC manufacturing process to ensure optimal product performance and reproducibility. SCTi003-A is karyotypically stable, demonstrates trilineage differentiation potential, expresses markers of the undifferentiated state, and was reprogrammed using a non-integrating reprogramming technology. The cell line is used as a healthy control for a multitude of pluripotent stem cell research applications including downstream differentiation to lineage-specific cell types and organoids.
Extensive quality control procedures are implemented at every stage of STEMCELL’s iPSC manufacturing process (Table 1). Our commercial iPSC quality assessments and release criteria have been developed based on recommendations and guidance from the Standards for Human Stem Cell Use In Research (ISSCR, 2023).
SCTi003-A is manufactured with mTeSR? Plus (Catalog no. 100-0276) and is fully compatible with STEMdiff? cell culture media products, allowing for standardized high-quality maintenance and differentiation to various cell types such as cardiomyocytes, neurons, astrocytes, and microglia.
SCTi003-A was derived from an αβ T cell and has undergone VDJ rearrangement.
The SCTi003-A certificate of analysis (COA) includes information relating to the product, cell line, recommended culture conditions, donor information, and detailed results for morphology, viability and recovery, cell line identity, sterility testing, mycoplasma testing, viral screening, parent cell lineage determination, chromosome analysis, 20q status, copy number variants, donor ancestry, genetic variants, TP53 and BCOR status, undifferentiated status, and pluripotency.
As an example, the certificate of analysis for SCTi003-A Lot # 2205404000 can be found here.
To learn more about SCTi003-A on the hPSCreg? website, click here.
Note - iPSC-Derived Forebrain Neuron Precursor Cells were manufactured from the SCTi003-A working cell bank (WCB).
Table 1. Characterization Assessments Are Performed at Various Stages throughout the iPSC Manufacturing Process. Master cell banks are tested for identity, adventitious agents, genomic integrity and stability, survival, undifferentiated state, and pluripotency. Working cell banks and commercial vials are tested for a subset of these characterization criteria.
Donor Information
STEMCELL collects donor demographic information ethically, using consent forms and protocols approved by either an Institutional Review Board (IRB), the Food and Drug Administration (FDA), the U.S. Department of Health and Human Services, and/or an equivalent regulatory authority. Donations are performed in the United States in compliance with applicable federal, state, and local laws, regulations, and guidance. Healthy donors must be over the age of 18, weigh at least 120 lb, have a body mass index (BMI) between 18.5–24.9, demonstrate no use of tobacco products, and be in good general health. Additionally, donors in our healthy pool are pre-screened using a health questionnaire aimed at excluding any donors with diseases, blood disorders, or other health concerns.
Table 2 details attributes that were determined for the SCTi003-A donor. Age, diagnosis, ethnicity and/or race, and tobacco use were self-declared by the SCTi003-A donor. Sex, ancestry, height, weight, BMI, blood type, HLA haplotype, and pathogenic genetic variants were calculated using various methods detailed in the table legend.
Table 2. iPSC Line SCTi003-A Is Derived from a Healthy Female Donor. Demographic, health, and genetic characteristics of the SCTi003-A donor were compiled based on self-reported information and whole-exome sequencing. Sex was determined by karyotype. Ancestry was calculated by EthSEQ analysis from whole-exome sequencing data. HLA haplotype was determined by next-generation sequencing, sequence-base typing, and sequence-specific oligonucleotide probes as needed to obtain the required resolution. Other genetic variants were determined from whole-exome sequencing using ClinVar analysis. Blood type (ABO/Rh blood group) was determined by next-generation sequencing. Height, weight, and BMI were calculated at the donation facility.
Forebrain Neuron Morphology
Morphologically, forebrain neuron precursor cells exhibit characteristics indicative of their developmental stage just prior to terminal differentiation into mature neurons. These cells typically display a small, round morphology with a high nuclear-to-cytoplasmic ratio, reflective of their precursor state. Upon thawing and plating, they quickly extend neurites and adopt a more complex, neuron-like morphology within 24 hours in culture. The precursors are cultured under defined conditions that promote the development of a mix of excitatory and inhibitory neurons, resulting in a robust and physiologically relevant model for forebrain neuron studies. Figure 1 demonstrates the high-quality morphology of iPSC-derived forebrain neuron precursor cells, showing their readiness to mature into functional forebrain neurons upon introduction to STEMdiff? Forebrain Neuron Maturation Medium (Catalog #08605).
Figure 1. Human iPSC-Derived Forebrain Neuron Precursor Cells Exhibit High-Quality Morphology. Cryopreserved Human iPSC-Derived Forebrain Neuron Precursor Cells, generated from SCTi003-A iPSCs, were thawed and plated onto PLO/Laminin-coated plates at a density of 80,000 cells/cm2 using the STEMdiff? Forebrain Neuron Maturation Kit. The neurons were incubated at 37°C and subsequently analyzed by brightfield microscopy at 10x magnification. Forebrain neurons displayed the expected neuronal morphology with minimal clumping over long-term culture.
Forebrain Neuron Marker Expression and Analysis
Marker expression in forebrain neurons can vary depending on the stage of differentiation. Typically, mature forebrain neurons express high levels of neuronal markers such as class III β-tubulin (βIII-TUB) while showing low expression of glial markers such as S100B. This distinct marker expression profile is indicative of their advanced neuronal differentiation status.
Human iPSC-derived forebrain neuron precursor cells were thawed, established in culture, and fixed for immunocytochemistry analysis. On Day 14, these neurons exhibited high expression of the neuronal marker βIII-TUB, with over 95% of the cells staining positive (Figure 2). Conversely, the glial marker S100B was expressed in less than 4% of the cells, highlighting the purity of the neuronal population. These results demonstrate that the differentiation protocol and culture conditions effectively promote the generation of a highly pure population of forebrain neurons. The high expression of βIII-TUB and low expression of S100B confirm the neuronal identity and maturity of these cells, making them suitable for various research applications.
领英推荐
Figure 2. Neurons Matured from Human iPSC-Derived Forebrain Neuron Precursor Cells Consist of a Highly Pure Neuronal Population. Human iPSC-Derived Neuron Precursor Cells, generated from SCTi003-A iPSCs, were thawed, established in culture, and fixed for immunocytochemistry. On Day 14, neurons expressed high levels of the neuronal marker βIII-TUB (red) with low expression of S100B (green). Additionally, they displayed typical neuronal morphology with healthy neurites and minimal cell clumping. (B) The percentage expression of these markers was quantified. The neuronal marker βIII-TUB was found to be expressed in 95% of neurons, while the glial marker S100B was expressed in less than 4% of neurons. Error bars represent standard deviation (n = 2 biological replicates).
Forebrain neurons exhibit a distinct marker expression pattern that reflects their mature neuronal status and forebrain-specific identity. These neurons typically express high levels of neuronal markers such as microtubule-associated protein 2 (MAP2), indicative of their mature neuronal phenotype. Additionally, they express markers that define their neurotransmitter identity and synaptic functionality.
Human iPSC-derived forebrain neuron precursor cells were thawed, cultured, and analyzed for marker expression using immunocytochemistry on Day 24. The neurons expressed the pan-neuronal marker MAP2, confirming their neuronal identity. They also expressed GABA, a marker for inhibitory neurons, indicating the presence of a substantial population of GABAergic neurons within the culture (Figure 3A). Furthermore, the forebrain-specific marker FOXG1 was prominently expressed, underscoring the regional identity of these neurons as forebrain neurons (Figure 3B). The expression of the pre-synaptic marker Synapsin further validated the functional maturation of these neurons, highlighting their capability for synaptic transmission (Figure 3C).
These results illustrate that the differentiated forebrain neurons display a healthy neuronal morphology with extensive neurite outgrowth and minimal cell clumping. The high expression levels of MAP2, GABA, FOXG1, and Synapsin indicate that these neurons are not only region-specific but also functionally active, making them highly suitable for various applications such as modeling neurological diseases, drug screening, and neurotoxicity testing.
Figure 3. Neurons Matured from Human iPSC-Derived Forebrain Neuron Precursor Cells Express Characteristic Neuronal Markers. Human iPSC-Derived Neuron Precursor Cells, generated from SCTi003-A iPSCs, were thawed, established in culture, and fixed for immunocytochemistry. On Day 24, neurons expressed neuronal marker MAP2, (A) GABA, (B) forebrain neuron marker FOXG1, and (C) pre-synaptic marker Synapsin. Additionally, cells displayed typical neuronal morphology with healthy neurites and minimal clumping.
The differentiation and maturation of forebrain neurons can be monitored through the expression of specific neuronal markers. RNA-seq analysis provides a comprehensive view of gene expression changes during the maturation process, offering insights into the developmental trajectory and functional maturation of these neurons.
Human iPSC-derived forebrain neuron precursor cells were matured using the STEMdiff? Forebrain Neuron Maturation Kit (Catalog #08605). RNA was harvested at various time points during maturation (Day 0, Day 14, and Day 21 in maturation media). The heat map (Figure 4) displays the expression levels of select genes associated with hPSCs, neural progenitor cells (NPCs), forebrain neuron precursors, and mature forebrain neurons.
At the hPSC stage, markers such as NANOG and OCT4 are highly expressed, indicative of their undifferentiated state. As differentiation progresses, the expression of NPC/forebrain neuron precursor markers such as PAX6, SOX1, SOX9, and POU3F2 increases, signifying the transition towards a neural lineage.
Upon maturation into forebrain neurons, there is a marked increase in the expression of neuronal markers. Genes such as TUBB3 (βIII-tubulin), GABRA1 (GABA receptor subunit), GRIA1-4 (glutamate receptor subunits), and GRIN2A/B (NMDA receptor subunits) become highly expressed, indicating the development of both excitatory and inhibitory neurons. Additional forebrain-specific markers such as FOXG1, TBR1, and ISL1 also show increased expression, confirming the regional identity of these neurons.
Markers indicative of mature neuronal function, including CAMK2A (calcium/calmodulin-dependent protein kinase II), SYN1 (synapsin I), and DLG4 (postsynaptic density protein 95), show significant upregulation by Day 21, reflecting the functional maturation of the neurons. The presence of GAD1 and GAD2 (glutamate decarboxylase 1 and 2) further confirms the presence of GABAergic neurons within the culture.
This gene expression profile demonstrates the successful differentiation and maturation of forebrain neurons from iPSC-derived precursors, providing a robust and physiologically relevant model for neuroscience research. These neurons exhibit key characteristics of forebrain neurons, making them highly suitable for studies on neurological development, disease modeling, and therapeutic screening.
Figure 4. Human iPSC-Derived Forebrain Neuron Precursor Cells Express Expected Neuronal Markers. Human iPSC-Derived Forebrain Neuron Precursor Cells were generated from the iPS cell line SCTi003-A. Neuron precursors were then matured using the STEMdiff? Forebrain Neuron Maturation Kit. RNA from the forebrain neurons was harvested at various time points during maturation (Day 0, Day 14, and Day 21 in maturation media). This RNA, along with RNA from the parental control line, was subsequently sequenced using bulk RNA-seq. The heat map displays expression levels for select genes associated with hPSCs, NPCs or forebrain neuron precursors, and forebrain neurons.
Electrophysiological Activity and Network Maturation of Forebrain Neurons
The functional maturation of forebrain neurons can be assessed by monitoring their electrophysiological activity over time. This analysis provides valuable insights into the development of neuronal networks and their capacity for synaptic transmission and synchronization. This work was performed in collaboration with Axion BioSystems.
Experimental Setup:
Human iPSC-derived Forebrain Neuron Precursor Cells were plated into an Axion CytoView MEA 48-well plate (Axion Catalog #M768-tMEA-48B), which features 16 electrodes per well. The cells were seeded at a density of 40,000 cells per well in a 5 μL droplet over the array, resulting in approximately 6 × 10? cells/cm2. The cells were maintained in STEMdiff? Forebrain Neuron Maturation Medium (Catalog #08605) for the duration of the 42-day recording period.
Electrophysiological Recordings:
The electrophysiological activity of the forebrain neurons was monitored using Axion's Maestro system. Recordings were taken at various time points (Day 1, Day 14, Day 28, and Day 42) to track the development of neuronal activity and network maturation. For longitudinal data, n=32 wells were recorded per time point.
As shown in Figure 5, the electrophysiological recordings demonstrated a progressive increase in neuronal activity as the cells matured. At Day 1, no significant activity was detected, indicating the initial stage of culture establishment. By Day 14, neurons began to exhibit sporadic firing events, reflecting the onset of network formation. This activity further increased by Day 28, with more frequent and organized firing patterns observed. By Day 42, the neurons demonstrated robust and synchronized firing, indicative of mature and well-established neuronal networks. The electrophysiological traces showed clear burst patterns and network-wide synchrony, highlighting the advanced functional state of the neurons.
Quantitative Analysis:
Conclusion:
The electrophysiological activity and network maturation data demonstrate that human iPSC-derived forebrain neurons develop robust and synchronized network activity over a 42-day period. These findings highlight the suitability of these neurons for studies on synaptic function, network dynamics, and neurological disease modeling. The ability to monitor and quantify electrophysiological parameters provides researchers with a powerful tool to assess neuronal functionality and network maturation in vitro.
Figure 5. Electrophysiological Activity and Network Maturation of Forebrain Neurons. Human iPSC-derived forebrain neuron precursor cells were seeded onto an Axion CytoView MEA 48-well plate (Axion Catalog #M768-tMEA-48B) and maintained in STEMdiff? Forebrain Neuron Maturation Medium (STEMCELL Catalog #08605). Electrophysiological activity was recorded at Days 1, 14, 28, and 42 using Axion's Maestro system. Top panels: Electrophysiological traces showing increased neuronal activity and synchronization over time. Bottom panels: Quantitative analysis of mean firing rate, number of bursts, and synchrony index, demonstrating progressive network maturation and increased activity. Data represent mean ± standard deviation from 32 wells per time point.
Summary
STEMCELL Technologies has developed cryopreserved Human iPSC-Derived Forebrain Neuron Precursor Cells (Catalog #200-0770) for academic and commercial research, optimized for functionality and broad applicability in studies of the human nervous system.
Human iPSC-Derived Forebrain Neuron Precursor Cells are available now at a price of $649 USD per vial (1 million viable cells). Unlike iPSC lines from STEMCELL's iPSC Repository, an annual license fee is not required for the use of our forebrain neurons.
For more information about STEMCELL's iPSC lines and differentiated cells, refer to our Frequently Asked Questions on iPSCs.
For any other queries, click here to contact STEMCELL's iPSC Team or email us directly at [email protected].
Senior Scientist @ RevivalBio / Stem Cell Development / Disease Modeling & Pharmacological Studies / Preclinical Trials
8 个月Very informative!
I have worked on iPSC-derived forebrain motor neurons, which are fascinating cells to study.