Stem Cell: Advanced Research and Therapy (ISSN: 2637-9589)

Article / research article

"A Novel Method for the Generation of Region-Specific Neurons and Neural Networks from Human Pluripotent Stem Cells"

Aynun N Begum1 and Yiling Hong1,2*
1College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA, USA
2Graduate College of Biomedical Sciences, Western University of Health Sciences, Pomona, CA, USA
*Corresponding author: Yiling HongCollege of Veterinary Medicine, Western University of Health Sciences, 309 East Second Street, Pomona, CA 91766-1854, USA, Tel: +1 9094698685; E-mail:
Received Date: 3 March, 2014;  Accepted Date: 7 April, 2016;  Published Date: 21 April, 2016  

Stem cell-based neuronal differentiation has provided a unique opportunity for disease modeling and regenerative medicine. We have reported a novel culture condition and method for generating neuronal progenitors and neural networks from human embryonic and induced pluripotent stem cells without any genetic manipulation. Neurospheres generated under 10% COwith Supplemented Knockout Serum Replacement Medium (SKSRM) had doubled the expression of NESTIN, PAX6 and FOXG1 genes compared to the neurospheres generated under 5% CO2. Furthermore, an additional step (AdStep) was introduced to fragment the neurospheres, which increased the expression of neuronal progenitor genes NEUROD1, NEUROG2, TBR1, TBR2, and NOTCH1 and the formation of the neuroepithelial-type cells. With the supplements, neuronal progenitors further differentiated into different layers of cortical, pyramidal, GABAergic, glutamatergic, cholinergic, dopaminergic and purkinje neurons within 27-40 days, which is faster than traditional neurodifferentiation protocols (42-60 days). Furthermore, our in vivo studies indicated that neuronal progenitors derived under our culture conditions with “AdStep” showed significantly increased neurogenesis in Severe Combined Immunodeficiency (SCID) mouse brains. This neurosphere-based neurodifferentiation protocol is a valuable tool for studies neurogenesis, neuronal transplantation and high throughput screening assays.

Stem cell based neuronal differentiation has provided the tools to generate large populations of neurons for “disease-in-a-dish” for neurological disorder research and regenerative medicine. There are two basic methods for generating neurons from human embryonic and induced Pluripotent Stem Cells (h/iPSCs): adherent (neuroectoderm) and non-adherent (embryoid body or neurosphere) culture conditions. Neuroectoderm use dual inhibition of SMAD signaling to promote efficient neuronal differentiation [1-4]. Although neuroectoderm is a well established method, utilizing neurospheres has many advantages over neuroectoderm. Neurospheres can be maintained and expanded without losing the expression of neural progenitor markers for more than 10 passages [5-7]. Furthermore, reports suggest that neurospheres derived from iPSCs with disease-specific genetic backgrounds can be used for many disease modeling studies, such as Timothy syndrome, Fragile X syndrome or Niemann Pick Disease (NPD), Alzheimer’s Disease (AD), Parkinson’s Disease (PD) [8-11]. Recently, we reported a new defined culture medium and conditions, namely, SKSRM medium and 10% CO2, for deriving neurospheres. Use of 10% CO2 culture conditions which increased the expression of NESTIN, PAX6, and FOXG1 compared to the traditional 5% CO2 culture conditions [12].

Relatively few studies describe the function of CO2 on neurogenesis. The physiological CO2 concentration in many tissues is far greater than that in which cells are typically cultured in vitro. This may influence the proliferation and differentiation potential of many cell types. Neurogenesis is also tightly regulated via both extrinsic environmental influences and intrinsic genetic factors. A large number of transcription factors have been implicated in the proliferation of neural progenitors, which ultimately influence the number of neurons in the developing brain [13,14]. Studies suggest that PAX6, Tbr1, Tbr2, and NeuroD are expressed sequentially during neurogenesis. They regulate diverse developmental mechanisms including regional identity, neuronal fate, cell cycle kinetics, cell migration, cell adhesion, axonal growth and guidance [15-17]. We investigated the influence of CO2 concentration on neurogenesis by comparing neurospheres derived from human Embryonic Stem Cells (hESCs) under two different culture conditions. The results showed that round and bright-edged neurospheres were generated in supplemented SKSRM with 10% CO2. In addition, the neurospheres generated under 10% CO2 were more stable, with double the expression of NESTIN, PAX6 and FOXG1 genes compared to those cultured with 5% CO2. However, the molecular mechanism of CO2 on controlling neurogenesis is still not clear. As previously reported, this could be due to reduced oxygenation or hypoxia [18]. There are several groups that have reported that hypoxia or reduced oxygenation enhances neural stem cell colony survival and increased NESTIN, SOX1, SOX2, and FOXG1 expression [19,20]. CO2 concentration appears to could be a fundamental factor in recreating the micro-environmental niche of the developing brain by altering the expression of key signaling proteins during early time-points of induced differentiation. Further investigation is needed to fully understand the molecular mechanisms of CO2 control of neurogenesis.

Neurospheres are a good source of neural progenitors for neural transplantation due to their easy delivery and ability to migrate [2,5]. However, clumping has been a challenge for neurodifferentiation both in vivo and in vitro, which results in less efficiency and a longer time period to generate subtype and region-specific functional neurons [7,21]. A procedure termed “AdStep” was introduced to broke neurospheres into smaller fragments which increased the efficiency of neuronal production. Neurospheres were collected and resuspended in the dissociation solution (Stem Cell Technologies), and then use 5 ml polystyrene serological pipette mechanical broken down into smaller fragments by 20-30 pounding motions. The pellet was collected and transferred to matrigel-coated plates. The monolayer neuroepithelium formed after 3-5 days cultures at 5% CO2, 37°C culture condition [12]. Our gene expression study result indicated that the “AdStep” increased the expression of NEUROD1, NEUROG2, TBR1, TBR2, MYT1, and NOTCH1. When compared to other current available neurodifferentiation methods, this procedure has allowed us to increase the number of neuroprogenitor cells and also enabled us to rapidly generate sub-type or region-specific neurons and neural networks. Using this protocol, cortical, glutamatergic, cholinergic, and GABAergic neurons were generated within 27 days. Supplementing with Sonic Hedgehog (SHH) and Fibroblast Growth Factor 8 (FGF8) allowed us to generate mid/hindbrain dopaminergic neurons and cerebellar purkinje neurons as well. In addition, the neurons derived from our protocol have strong ongoing spontaneous Ca2+ activities as measured by Fluo-4, Ca2+ indicator dye. Time lapse Ca2+ imaging demonstrated that neurons derived from neuropsheres with AdStep have higher networking neurons compared to the neuroectoderm derived neurons [22].

We then injected the AdStep-fragmented and the non-fragmented neurospheres into Severe Combined Immunodeficiency (SCID) mouse brains to investigate the effect of “AdStep” on neurogenesis in vivo. Six weeks after neurosphere transplantation, the mice were sacrificed and histological analysis was conducted with antibodies against Human-Specific Nuclear Antigen (HumN) and βIII-Tubulin in order to identify the transplanted human cells. The results suggested that the engrafted AdStep-fragmented neurospheres differentiated into multipotent neural stem cells and mature neurons in vivo at a higher amount compared to the non-fragmented neurospheres. It is important to note that these results may have significant impacts on neuronal transplantation and regenerative medicine.

In conclusion, this novel and viral-free method for rapidly generating neurons from neurospheres provides a valuable model system for studying neurogenesis and understanding the molecular mechanisms associated with neurodegenerative diseases. This neurodifferentiation method is an excellent protocol for transplantation studies of various neurological disorders and regenerative medicine.


This work was supported by the National Institutes of Environmental Health Sciences (1R15 ES019298-01A1).

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Citation: Begum AN, Hong Y (2016) A Novel Method for the Generation of Region-Specific Neurons and Neural Networks from Human Pluripotent Stem Cells. Gavin J Stem Cell Res Ther 2016: 1-3.

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