Accepted Manuscript Title: Production of Neural Stem Cells from Human Pluripotent Stem Cells Author: Yu Wen Sha Jin PII: DOI: Reference:
S0168-1656(14)00802-5 http://dx.doi.org/doi:10.1016/j.jbiotec.2014.07.453 BIOTEC 6805
To appear in:
Journal of Biotechnology
Received date: Revised date: Accepted date:
8-4-2014 1-7-2014 31-7-2014
Please cite this article as: Wen, Y., Jin, S.,Production of Neural Stem Cells from Human Pluripotent Stem Cells, Journal of Biotechnology (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.07.453 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Production of Neural Stem Cells from Human Pluripotent Stem Cells
1
ip t
Yu Wen1,3, Sha Jin1, 2*
Department of Biomedical Engineering, College of Engineering, University of Arkansas,
2
cr
Fayetteville, AR 72701, USA
Department of Bioengineering, Thomas J. Watson School of Engineering and Applied
us
Sciences, State University of New York in Binghamton, Binghamton, New York 13902,
3
an
USA
Department of Histology and Embryology, School of Basic Medicine, China Medical
d
M
University, Shenyang 110001, P.R. China
te
*To whom correspondence should be addressed: Department of Bioengineering, Thomas J. Watson School of Engineering and Applied Sciences, SUNY Binghamton, NY 13902, USA.
Ac ce p
Phone: 1-607-777-5082. Fax: 1-607-777-5780. Email: [email protected]
Abstract
Despite significant advances in commercially available media and kits and the differentiation approaches for human neural stem cell (NSC) generation, NSC production from the differentiation of human pluripotent stem cell (hPSC) is complicated by its time-consuming procedure, complex medium composition, and purification step. In this study, we developed a convenient and simplified NSC production protocol to meet the demand of NSC production. We demonstrated that NSCs can be generated efficiently without requirement of specific small molecules or embryoid body formation stage. Our experimental results suggest that a
-1Page 1 of 29
short suspension culture period may facilitate ectoderm lineage specification rather than endoderm or mesoderm lineage specification from hPSCs. The method developed in this study shortens the turnaround time of NSC production from both human embryonic stem
ip t
cells (hESCs) and induced pluripotent stem cells (iPSCs) differentiation. It provides a straightforward and useful strategy for generating NSCs that can benefit a wide range of
an
us
cr
research applications for human brain research.
Keywords: human neural stem cells, differentiation, induced pluripotent stem cells, human
Ac ce p
te
d
M
embryonic stem cells, neural rosette, suspension culture.
-2Page 2 of 29
1. Introduction Brain research has long been harshly hindered by a lack of available human brain tissues. One of the major facts is that unlike most organs, where tissue samples can be taken via
ip t
biopsy, biopsy is not adapted for the human brain. Thus, a majority of researches in the field of neurodevelopmental disorders have to rely on small animal studies. Unfortunately, it is
cr
often found that experimental results obtained from small animals do not represent the real situation that occurs in humans (Amit et al., 2000; Hong et al., 2008). Recently, human
us
pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced
an
pluripotent stem cells (iPSCs), became an excellent renewable source of brain tissue, as they can be used to produce neurons and glia subtypes by using advanced differentiation
M
procedures. For instance, human neural stem cells (NSCs) differentiated from hPSCs may serve as an influential tool for modelling brain development and neurological diseases. In
d
particular, patient-specific NSCs can be generated by taking advantage of iPSCs technology.
te
Therefore, efficient production and expansion of NSCs from hPSCs and further differentiation into neural tissue are prerequisites for cell-based replacement therapies and
Ac ce p
new drug screening of the nervous system.
Extensive efforts have been made for developing protocol for effective human NSC generation from differentiation of hPSC to date. Usually, a minimum of four to seven days of embryoid body (EB) formation stage as an initial step of a stepwise NSC generation procedure is reported (Falk et al., 2012; Nemati et al., 2011; Yuan et al., 2011). Yuan and his co-workers isolated a population of NSCs that was CD184+/CD271-/CD44-/CD24+ from both of hPSCs after four days of EB formation stage and sixteen days of adherent culture on Matrigel-coated surface (Yuan et al., 2011). Some procedures even require a longer time period for the EB culture to produce NSCs. For instance, Liu group reported generating NSCs
-3Page 3 of 29
from hPSCs by initiating ten days of EB culture in a complicated medium, including conditioned medium of a stromal cell line PA6, Rock inhibitor, growth factors, followed by four to seven days of adherent culture before cell sorting(Liu et al., 2012). hESCs seeded in a
ip t
monolayer culture environment took approximately one month to generate the neural rosettes (Zhao et al., 2012). Very recently, neural differentiation media and kits have become
cr
commercially available despite the high prices (Elkabetz et al., 2008; Elkabetz and Studer, 2008; Hong et al., 2008; Koch et al., 2009; Nemati et al., 2011; Yan et al., 2013). As an
us
alternative, methods have been developed for direct lineage specification of hPSCs into a
an
specific neural cell type, including dopaminergic neuron, motor neuron, astrocyte, and oligodendrocyte (Amoroso et al., 2013; Cai et al., 2013; Liu et al., 2013; Liu et al., 2011;
M
Schwartz et al., 2012; Shaltouki et al., 2013).
d
Another strategy is to find small-molecules to leverage intracellular signalling pathways for
te
promoting NSC differentiation efficiency (Falk et al., 2012; Koch et al., 2009; Li et al., 2011). There are a number of small molecules that can be supplemented into the NSC
Ac ce p
differentiation medium for inducing NSC generation. These molecules include but are not limited to dorsomorphin, CHIR99021, SB431542, insulin, transferrin, sodiumselenite, fibronectin, noggin, selenous acid, EGF, and bFGF (Falk et al., 2012; Koch et al., 2009; Li et al., 2011; Yuan et al., 2011). Direct generation of neurons from hPSC using multiple small molecules avoids completed procedures for NSC production (Chambers et al., 2012; Li et al., 2011). Inhibitors that can suppress bone morphogenic protein and TGFβ/activin/nodal signaling promote neural lineage specification (Morizane et al., 2011). Notch related transmembrane protein Dlk1 can promote the differentiation of hESC-derived neural progenitors via Notch signalling (Surmacz et al., 2012). Hence, the addition of Dlk1 can significantly enhance neural progenitor production. Simultaneously use of two inhibitors of
-4Page 4 of 29
SMAD signaling can facilitate neural conversion of hPSCs (Chambers et al., 2009). Based on the knowledge that human primitive neural precursors can be generated from hESCs if glycogen synthase kinase 3(GSK3), transforming growth factor β (TGF-β), and Notch
ip t
pathways are inhibited, a combination of multiple small molecules can direct cell lineage specification from hESCs without EB formation stage (Li et al., 2011). Nevertheless, most of
cr
these differentiation procedures were complicated by either utilizing multiple small molecules and growth factors or requiring EB formation as the first step of lineage restriction,
us
followed by tedious mechanical selection of neuroepithelial precursors to mimic early human
an
embryogenesis. Moreover, many studies have utilized the mechanical isolation of neural rosettes, which is inefficient and leads to a heterogeneous population of neural cells
M
containing undefined derivative and remnants of undifferentiated hPSCs. For example, Ebert group developed a method to generate and expand pre-rosette neural progenitors by
d
suspension culture of hESCs and iPSCs in a differentiation medium containing heparin and
te
high concentration of EGF and bFGF . Cell aggregates were passaged using an automated tissue chopping apparatus manufactured by Mickle Laboratory Engineering Co. Ltd in United
Ac ce p
Kindom (Ebert et al., 2013; Svendsen et al., 1998). However, the automated tissue chopping technique requires specific equipment and the device fails to selectively passage pro-rosettes that have the potential to be NSCs or neural progenitor cells (NPCs). Hence, it is necessary to develop a simplified NSC generation protocol to meet the demand of studies in the fields of brain development and neurological diseases.
In this study, we describe our recent findings that short period of suspension culture may facilitate ectoderm lineage specification from hPSCs. NSCs can be generated from hPSCs using a straightforward and cost-effective culture medium without the special need of signalling molecules and an EB formation step. This new approach is also time-effective, and
-5Page 5 of 29
allows for rapid production of NSCs, representing a forthright strategy for generating selfrenewable NSCs that can facilitate a wide range of scientific research applications for human
ip t
brain researches.
2. Materials and Methods hESC and iPSC culture
cr
2.1.
The hESC line H9 and iPSC line IMR90 (WiCell Institute) were maintained in
us
undifferentiated state on growth factor-reduced Matrigel (MG)-coated (BD Biosciences)
an
dishes in an mTeSR1 (StemCell Technologies) medium at 37°C and 5% CO2 atmosphere as described in our previous studies (Jin et al., 2012a; Jin et al., 2012b). Cells were fed with new
M
medium every day. For the subculture, hESCs and hiPSCs were treated with 1mg/mL dispase (StemCell Technologies) for 7 min at 37°C, and rinsed with Dulbecco’s Modified Eagle
d
Medium (DMEM)/F12 three times. The cell colonies were gently scraped from the dishes and
te
pipetted several times to break the colonies up into small pieces before re-plating. Cells were
Ac ce p
split at a ratio of 1:3 every 3-4 days.
2.2.
Neural Rosette formation
Undifferentiated H9 and IMR90 cell colonies were detached using 0.5 mg/mL dispasecontaining DMEM/F12 for 30 min at 37°C when the cell colony density reached 70-80% confluence. The cells were then transferred to a 15 ml conical tube and allowed to sink to the bottom of the tube before carefully aspirating the medium. After washing cells with DMEM/F12 medium three times, the cells were resuspended in a proper amount of mTeSR1 to allow the cell density to reach approximately 1x105 cells/mL. The cells were gently pipetted up and down ten times and then seeded to a cell culture plate, after which they were cultured at 37°C 5% CO2 and 95% air for 24 h. The cell aggregates were collected and
-6Page 6 of 29
replated to 80µg/mL Matrigel and 10mg/mL poly-L-ornithine (Sigma) coated plate and cultured in NSC medium containing DMEM/F12, 2% B27, 2 mM L-glutamine, 20 ng/ml EGF, and 20 ng/ml bFGF. The NSC medium was replaced every other day. Neural rosettes
ip t
appeared in 3~4 days and the number of neural rosettes kept growing with further culturing for another 2~3 days. In the EB formation method, cells were resuspended in mTESR1
cr
medium after dispase treatment as described above. Cells were then seeded to an ultra-low attachment plate (Corning) and cultured in an mTeSR1 medium for 4~7 days after up and
us
down pipetting. Half of the culture medium was changed every day. The formation of neural
an
rosette was examined under a phase contrast microscope. The number of rosettes was counted
2.3.
M
at various microscopic fields by randomly selecting five fields for each sample.
Isolation of neural rosettes for NSC generation
d
Neural rosettes were treated with Neural Rosette Selection Reagent (StemCell Technologies)
te
for 1 h at 37°C. The reagent was removed carefully from the culture plate, and cell clusters containing neural rosettes were lifted off and collected to a DMEM/F12 medium. The rosette
Ac ce p
clusters were then transferred to a 15ml tube and centrifuged for 5 min at 300 x g. The rosettes were then resuspended in NSC medium and seeded onto 10mg/mL poly-L-ornithine and 1mg/mL Laminin coated 6-well plate. The medium was changed every other day, and the cells were cultured until they reached 80-90% confluence before passaging. For passaging NSCs, cells were treated with Accutase and then subcultured. The cells were passaged once per week at a split ratio of 1:3. The schematic diagram of the approach for efficient NSC production developed in this study is summarized in Fig. 1A.
2.4.
Generation of neuronal cells from NSC differentiation
-7Page 7 of 29
NSCs after 3~4 passages were transferred to a low attachment plate to allow the suspension culture in neuronal medium containing DMEM/F12, 1% fetal bovine serum (ATCC), 2% B27, and 2 mM L-glutamine. Neurospheres appeared after 2~3 weeks. They were seeded
ip t
onto poly-L-ornithine and laminin-coated plate and cultured in the neuronal medium for another 2~3 weeks. The same medium was used for glial cell and oligodendrocyte
2.5.
us
cr
differentiation from NSC.
Gene expression assay
an
To detect gene expression, total RNA was isolated from cells after eliminating genomic DNA using an RNA extraction kit from Qiagen. The TaqMan qRT-PCR approach was applied as
M
described elsewhere(Jin et al., 2012a; Jin et al., 2012b). Cyclophilin (Invitrogen), a human endogenous gene, served as a housekeeping gene for normalization. To determine gene
d
expressions of pluripotent, NSC, and three germ layer markers, OCT4, SOX2, DACH1,
te
GBX2, PLZF, PAX6, Sox17, Hind1, and Nestin primer-probe sets were used (Table 1), as reported elsewhere(Jin et al., 2012a). The relative gene expression level was expressed as
Ac ce p
fold changes to the corresponding values detected from those obtained from existing EB formation methods or to undifferentiated cell status (Elkabetz et al., 2008; Falk et al., 2012). Control assays were performed to ensure the absence of genomic DNA contamination in the qRT-PCR assay.
2.6.
Immunofluorescence microscopy
A standard immunofluorescence staining protocol was used, as reported previously(Jin et al., 2012a; Jin et al., 2012b). To briefly describe, cells were fixed in a 4% paraformaldehyde in PBS and incubated in a blocking buffer containing 5% goat and monkey serum (Jackson ImmunoResearch) and 0.3% Triton X-100/PBS buffer for 60 min at room temperature. Cells
-8Page 8 of 29
were then incubated with diluted primary antibodies overnight at 4°C (Table 2). Cells were further incubated in secondary antibodies for 1~2 h at room temperature in the dark (Table 2). Finally, the samples were stained with DAPI (Vector Laboratories) for 15 min to visualize
ip t
the nuclei. Cell images were captured using Olympus IX-71 fluorescence microscope, as
2.7.
cr
described elsewhere(Jin et al., 2011).
Statistical Analysis
us
All experiments were carried out for at least three times independently. Student’s t-test was
an
used to statistically analyse and interpret the data. A p-value
S0168-1656(14)00802-5 http://dx.doi.org/doi:10.1016/j.jbiotec.2014.07.453 BIOTEC 6805
To appear in:
Journal of Biotechnology
Received date: Revised date: Accepted date:
8-4-2014 1-7-2014 31-7-2014
Please cite this article as: Wen, Y., Jin, S.,Production of Neural Stem Cells from Human Pluripotent Stem Cells, Journal of Biotechnology (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.07.453 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Production of Neural Stem Cells from Human Pluripotent Stem Cells
1
ip t
Yu Wen1,3, Sha Jin1, 2*
Department of Biomedical Engineering, College of Engineering, University of Arkansas,
2
cr
Fayetteville, AR 72701, USA
Department of Bioengineering, Thomas J. Watson School of Engineering and Applied
us
Sciences, State University of New York in Binghamton, Binghamton, New York 13902,
3
an
USA
Department of Histology and Embryology, School of Basic Medicine, China Medical
d
M
University, Shenyang 110001, P.R. China
te
*To whom correspondence should be addressed: Department of Bioengineering, Thomas J. Watson School of Engineering and Applied Sciences, SUNY Binghamton, NY 13902, USA.
Ac ce p
Phone: 1-607-777-5082. Fax: 1-607-777-5780. Email: [email protected]
Abstract
Despite significant advances in commercially available media and kits and the differentiation approaches for human neural stem cell (NSC) generation, NSC production from the differentiation of human pluripotent stem cell (hPSC) is complicated by its time-consuming procedure, complex medium composition, and purification step. In this study, we developed a convenient and simplified NSC production protocol to meet the demand of NSC production. We demonstrated that NSCs can be generated efficiently without requirement of specific small molecules or embryoid body formation stage. Our experimental results suggest that a
-1Page 1 of 29
short suspension culture period may facilitate ectoderm lineage specification rather than endoderm or mesoderm lineage specification from hPSCs. The method developed in this study shortens the turnaround time of NSC production from both human embryonic stem
ip t
cells (hESCs) and induced pluripotent stem cells (iPSCs) differentiation. It provides a straightforward and useful strategy for generating NSCs that can benefit a wide range of
an
us
cr
research applications for human brain research.
Keywords: human neural stem cells, differentiation, induced pluripotent stem cells, human
Ac ce p
te
d
M
embryonic stem cells, neural rosette, suspension culture.
-2Page 2 of 29
1. Introduction Brain research has long been harshly hindered by a lack of available human brain tissues. One of the major facts is that unlike most organs, where tissue samples can be taken via
ip t
biopsy, biopsy is not adapted for the human brain. Thus, a majority of researches in the field of neurodevelopmental disorders have to rely on small animal studies. Unfortunately, it is
cr
often found that experimental results obtained from small animals do not represent the real situation that occurs in humans (Amit et al., 2000; Hong et al., 2008). Recently, human
us
pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced
an
pluripotent stem cells (iPSCs), became an excellent renewable source of brain tissue, as they can be used to produce neurons and glia subtypes by using advanced differentiation
M
procedures. For instance, human neural stem cells (NSCs) differentiated from hPSCs may serve as an influential tool for modelling brain development and neurological diseases. In
d
particular, patient-specific NSCs can be generated by taking advantage of iPSCs technology.
te
Therefore, efficient production and expansion of NSCs from hPSCs and further differentiation into neural tissue are prerequisites for cell-based replacement therapies and
Ac ce p
new drug screening of the nervous system.
Extensive efforts have been made for developing protocol for effective human NSC generation from differentiation of hPSC to date. Usually, a minimum of four to seven days of embryoid body (EB) formation stage as an initial step of a stepwise NSC generation procedure is reported (Falk et al., 2012; Nemati et al., 2011; Yuan et al., 2011). Yuan and his co-workers isolated a population of NSCs that was CD184+/CD271-/CD44-/CD24+ from both of hPSCs after four days of EB formation stage and sixteen days of adherent culture on Matrigel-coated surface (Yuan et al., 2011). Some procedures even require a longer time period for the EB culture to produce NSCs. For instance, Liu group reported generating NSCs
-3Page 3 of 29
from hPSCs by initiating ten days of EB culture in a complicated medium, including conditioned medium of a stromal cell line PA6, Rock inhibitor, growth factors, followed by four to seven days of adherent culture before cell sorting(Liu et al., 2012). hESCs seeded in a
ip t
monolayer culture environment took approximately one month to generate the neural rosettes (Zhao et al., 2012). Very recently, neural differentiation media and kits have become
cr
commercially available despite the high prices (Elkabetz et al., 2008; Elkabetz and Studer, 2008; Hong et al., 2008; Koch et al., 2009; Nemati et al., 2011; Yan et al., 2013). As an
us
alternative, methods have been developed for direct lineage specification of hPSCs into a
an
specific neural cell type, including dopaminergic neuron, motor neuron, astrocyte, and oligodendrocyte (Amoroso et al., 2013; Cai et al., 2013; Liu et al., 2013; Liu et al., 2011;
M
Schwartz et al., 2012; Shaltouki et al., 2013).
d
Another strategy is to find small-molecules to leverage intracellular signalling pathways for
te
promoting NSC differentiation efficiency (Falk et al., 2012; Koch et al., 2009; Li et al., 2011). There are a number of small molecules that can be supplemented into the NSC
Ac ce p
differentiation medium for inducing NSC generation. These molecules include but are not limited to dorsomorphin, CHIR99021, SB431542, insulin, transferrin, sodiumselenite, fibronectin, noggin, selenous acid, EGF, and bFGF (Falk et al., 2012; Koch et al., 2009; Li et al., 2011; Yuan et al., 2011). Direct generation of neurons from hPSC using multiple small molecules avoids completed procedures for NSC production (Chambers et al., 2012; Li et al., 2011). Inhibitors that can suppress bone morphogenic protein and TGFβ/activin/nodal signaling promote neural lineage specification (Morizane et al., 2011). Notch related transmembrane protein Dlk1 can promote the differentiation of hESC-derived neural progenitors via Notch signalling (Surmacz et al., 2012). Hence, the addition of Dlk1 can significantly enhance neural progenitor production. Simultaneously use of two inhibitors of
-4Page 4 of 29
SMAD signaling can facilitate neural conversion of hPSCs (Chambers et al., 2009). Based on the knowledge that human primitive neural precursors can be generated from hESCs if glycogen synthase kinase 3(GSK3), transforming growth factor β (TGF-β), and Notch
ip t
pathways are inhibited, a combination of multiple small molecules can direct cell lineage specification from hESCs without EB formation stage (Li et al., 2011). Nevertheless, most of
cr
these differentiation procedures were complicated by either utilizing multiple small molecules and growth factors or requiring EB formation as the first step of lineage restriction,
us
followed by tedious mechanical selection of neuroepithelial precursors to mimic early human
an
embryogenesis. Moreover, many studies have utilized the mechanical isolation of neural rosettes, which is inefficient and leads to a heterogeneous population of neural cells
M
containing undefined derivative and remnants of undifferentiated hPSCs. For example, Ebert group developed a method to generate and expand pre-rosette neural progenitors by
d
suspension culture of hESCs and iPSCs in a differentiation medium containing heparin and
te
high concentration of EGF and bFGF . Cell aggregates were passaged using an automated tissue chopping apparatus manufactured by Mickle Laboratory Engineering Co. Ltd in United
Ac ce p
Kindom (Ebert et al., 2013; Svendsen et al., 1998). However, the automated tissue chopping technique requires specific equipment and the device fails to selectively passage pro-rosettes that have the potential to be NSCs or neural progenitor cells (NPCs). Hence, it is necessary to develop a simplified NSC generation protocol to meet the demand of studies in the fields of brain development and neurological diseases.
In this study, we describe our recent findings that short period of suspension culture may facilitate ectoderm lineage specification from hPSCs. NSCs can be generated from hPSCs using a straightforward and cost-effective culture medium without the special need of signalling molecules and an EB formation step. This new approach is also time-effective, and
-5Page 5 of 29
allows for rapid production of NSCs, representing a forthright strategy for generating selfrenewable NSCs that can facilitate a wide range of scientific research applications for human
ip t
brain researches.
2. Materials and Methods hESC and iPSC culture
cr
2.1.
The hESC line H9 and iPSC line IMR90 (WiCell Institute) were maintained in
us
undifferentiated state on growth factor-reduced Matrigel (MG)-coated (BD Biosciences)
an
dishes in an mTeSR1 (StemCell Technologies) medium at 37°C and 5% CO2 atmosphere as described in our previous studies (Jin et al., 2012a; Jin et al., 2012b). Cells were fed with new
M
medium every day. For the subculture, hESCs and hiPSCs were treated with 1mg/mL dispase (StemCell Technologies) for 7 min at 37°C, and rinsed with Dulbecco’s Modified Eagle
d
Medium (DMEM)/F12 three times. The cell colonies were gently scraped from the dishes and
te
pipetted several times to break the colonies up into small pieces before re-plating. Cells were
Ac ce p
split at a ratio of 1:3 every 3-4 days.
2.2.
Neural Rosette formation
Undifferentiated H9 and IMR90 cell colonies were detached using 0.5 mg/mL dispasecontaining DMEM/F12 for 30 min at 37°C when the cell colony density reached 70-80% confluence. The cells were then transferred to a 15 ml conical tube and allowed to sink to the bottom of the tube before carefully aspirating the medium. After washing cells with DMEM/F12 medium three times, the cells were resuspended in a proper amount of mTeSR1 to allow the cell density to reach approximately 1x105 cells/mL. The cells were gently pipetted up and down ten times and then seeded to a cell culture plate, after which they were cultured at 37°C 5% CO2 and 95% air for 24 h. The cell aggregates were collected and
-6Page 6 of 29
replated to 80µg/mL Matrigel and 10mg/mL poly-L-ornithine (Sigma) coated plate and cultured in NSC medium containing DMEM/F12, 2% B27, 2 mM L-glutamine, 20 ng/ml EGF, and 20 ng/ml bFGF. The NSC medium was replaced every other day. Neural rosettes
ip t
appeared in 3~4 days and the number of neural rosettes kept growing with further culturing for another 2~3 days. In the EB formation method, cells were resuspended in mTESR1
cr
medium after dispase treatment as described above. Cells were then seeded to an ultra-low attachment plate (Corning) and cultured in an mTeSR1 medium for 4~7 days after up and
us
down pipetting. Half of the culture medium was changed every day. The formation of neural
an
rosette was examined under a phase contrast microscope. The number of rosettes was counted
2.3.
M
at various microscopic fields by randomly selecting five fields for each sample.
Isolation of neural rosettes for NSC generation
d
Neural rosettes were treated with Neural Rosette Selection Reagent (StemCell Technologies)
te
for 1 h at 37°C. The reagent was removed carefully from the culture plate, and cell clusters containing neural rosettes were lifted off and collected to a DMEM/F12 medium. The rosette
Ac ce p
clusters were then transferred to a 15ml tube and centrifuged for 5 min at 300 x g. The rosettes were then resuspended in NSC medium and seeded onto 10mg/mL poly-L-ornithine and 1mg/mL Laminin coated 6-well plate. The medium was changed every other day, and the cells were cultured until they reached 80-90% confluence before passaging. For passaging NSCs, cells were treated with Accutase and then subcultured. The cells were passaged once per week at a split ratio of 1:3. The schematic diagram of the approach for efficient NSC production developed in this study is summarized in Fig. 1A.
2.4.
Generation of neuronal cells from NSC differentiation
-7Page 7 of 29
NSCs after 3~4 passages were transferred to a low attachment plate to allow the suspension culture in neuronal medium containing DMEM/F12, 1% fetal bovine serum (ATCC), 2% B27, and 2 mM L-glutamine. Neurospheres appeared after 2~3 weeks. They were seeded
ip t
onto poly-L-ornithine and laminin-coated plate and cultured in the neuronal medium for another 2~3 weeks. The same medium was used for glial cell and oligodendrocyte
2.5.
us
cr
differentiation from NSC.
Gene expression assay
an
To detect gene expression, total RNA was isolated from cells after eliminating genomic DNA using an RNA extraction kit from Qiagen. The TaqMan qRT-PCR approach was applied as
M
described elsewhere(Jin et al., 2012a; Jin et al., 2012b). Cyclophilin (Invitrogen), a human endogenous gene, served as a housekeeping gene for normalization. To determine gene
d
expressions of pluripotent, NSC, and three germ layer markers, OCT4, SOX2, DACH1,
te
GBX2, PLZF, PAX6, Sox17, Hind1, and Nestin primer-probe sets were used (Table 1), as reported elsewhere(Jin et al., 2012a). The relative gene expression level was expressed as
Ac ce p
fold changes to the corresponding values detected from those obtained from existing EB formation methods or to undifferentiated cell status (Elkabetz et al., 2008; Falk et al., 2012). Control assays were performed to ensure the absence of genomic DNA contamination in the qRT-PCR assay.
2.6.
Immunofluorescence microscopy
A standard immunofluorescence staining protocol was used, as reported previously(Jin et al., 2012a; Jin et al., 2012b). To briefly describe, cells were fixed in a 4% paraformaldehyde in PBS and incubated in a blocking buffer containing 5% goat and monkey serum (Jackson ImmunoResearch) and 0.3% Triton X-100/PBS buffer for 60 min at room temperature. Cells
-8Page 8 of 29
were then incubated with diluted primary antibodies overnight at 4°C (Table 2). Cells were further incubated in secondary antibodies for 1~2 h at room temperature in the dark (Table 2). Finally, the samples were stained with DAPI (Vector Laboratories) for 15 min to visualize
ip t
the nuclei. Cell images were captured using Olympus IX-71 fluorescence microscope, as
2.7.
cr
described elsewhere(Jin et al., 2011).
Statistical Analysis
us
All experiments were carried out for at least three times independently. Student’s t-test was
an
used to statistically analyse and interpret the data. A p-value
