Parkinson’s disease cell models—part 2 - Thermo Fisher Scientific · Parkinson’s disease (PD)...

Parkinson’s disease cell models—part 2Generation and characterization of neural stem cells and neurons from Parkinson’s disease donor-derived iPSC lines

Contents

5 What is Parkinson’s disease?

5 Collaboration summary

7 NSC overview

8 NSC generation

10 Cytogenetic analysis

11 Immunocytochemistry for NSC markers

12 Immunostaining and flow cytometry analysis for NSC markers

13 Cell line authentication

14 Gene expression profiling of NSCs

15 Differentiation to dopaminergic neurons

16 Differentiation to glial cell types

17 Gene expression profiling of differentiated NSCs

20 Functional assays to assess NSC health

23 Summary

NSC-derived DA neurons express dopaminergic marker TH (green) after 48 days of differentiation. Co-stained with Hoechst® 33342 DNA stain (blue) for cell nuclei.

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NSC-derived DA neurons express dopaminergic marker TH (green) and the neuronal marker β-III tubulin (red) after 48 days of differentiation. Co-stained with Hoechst® 33342 DNA stain (blue) for cell nuclei.

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Dopamine system

Frontal lobe

Ventral tegmental area

Striatum

Substantia nigra

Research summaryWhat is Parkinson’s disease?Parkinson’s disease (PD) is a progressive neurodegenerative disorder that affects 1% of people over age 60 and more than 5 million people worldwide [1]. PD results primarily from the selective loss of dopaminergic neurons in the substantia nigra region of the brain. The loss of these neurons first affects movement, but then later affects cognitive function, with late-stage disease often being accompanied by dementia. The absence of physiologically relevant cellular models for PD represents a major bottleneck for PD research. There is an urgent need for novel models that accelerate the discovery of disease mechanisms or drug targets, or for screening purposes that could accelerate the development of clinical and therapeutic applications. Patient-specific iPSC-derived cell types have become attractive tools for disease modeling in vitro.

Collaboration summaryRecently, Life Technologies partnered with the Parkinson’s Institute in Sunnyvale, California to develop PD model systems using donor fibroblasts that were collected at the Institute. The first step in creating the model systems was generation of induced pluripotent stem cells (iPSCs), which were then characterized using cellular and genetic analysis tools to confirm successful reprogramming. Here, we describe the second step in creating the model systems—the generation and characterization of neural stem cells (NSCs) from PD donor-derived iPSC lines, including differentiation of NSCs to dopaminergic neurons or other relevant cell types to study

the associated cell types involved in PD. This story is the second published in this series; future studies will describe genomic editing of specific PD-related genes using TAL-based or other technologies to study the impact of specific mutations within the genome, followed by development of assays to observe PD-relevant phenotypes within the resulting differentiated cells. With this work, we intend to demonstrate how systems can be developed to identify drugs that may ameliorate the processes that underlie PD, or to understand the environmental factors that impact the development of PD.


NSC overviewTraditionally, NSCs have been generated via embryoid body (EB) formation. In this method, EBs are first derived from iPSCs, followed by isolation and expansion of neural rosettes from the EBs, which adds time, labor, and variability. The EB approach generates NSCs that can differentiate into different neural cell types or be cryopreserved for further expansion, but this is an inefficient, laborious process that often results in a low yield of NSCs. To overcome these limitations, we developed a novel neural induction method that allows for the generation of NSCs from iPSCs within 7 days without the need for EB formation [2].

In the work presented here, we differentiated iPSC lines from three PD-affected donors (PD-1, PD-2, and PD-3), one multiple systems atrophy (MSA)–affected donor, and two age-matched, healthy control individuals (Ctrl-1 and Ctrl-2) into NSCs using this novel neural induction medium. MSA has been described as a more severe form of PD, with a more rapid disease progression after the first appearance of symptoms [3]. Fibroblasts from PD donors had previously been genotyped, and were known to contain LRRK2, GBA, and PARK2

mutations, whereas the MSA line was from a sporadic case with no mutations in genes known to be associated with PD.

We demonstrate that the generated NSCs express the known NSC markers Nestin, Sox1, and Sox2, and are karyotypically normal. Furthermore, gene expression analysis distinguishes these NSCs from their parental iPSCs and fibroblasts, and groups them with control NSCs derived from H9 embryonic stem cells (ESCs). We show that the derived NSCs can be used in functional assays to assess various aspects of neural stem cell health in response to cellular stressors, and finally, that they can be further differentiated into dopaminergic neurons and glial cells.

In summary, this novel neural induction medium allows efficient and robust generation of NSCs from PD donor–derived iPSCs. This method has potential utilization in large-scale NSC generation for high-throughput/high-content drug screening [4] and for further differentiating the NSCs into a variety of neural cell types for in vitro disease modeling.

• Three PD donors (PD-1, PD-2, and PD-3)

• One multiple systems atrophy (MSA) donor

• Two age-matched, non-diseased control individuals (Ctrl-1 and Ctrl-2)

Six iPSC cell lines from these donors were differentiated into NSCs:

8 Life Technologies | Parkinson’s cell models

Figure 1. Workflow for NSC generation and characterization.

1. Cytogenetic analysis.

2. Immunocytochemistry for Nestin, Sox1, Sox2, Pax6, and Oct4 (negative control).

3. Flow cytometry analysis for Nestin, Sox1, Sox2, Pax6, and Nanog (negative control).

4. Gene expression profiling.

5. Cell authentication.

6. Differentiation into dopaminergic neurons and glial astrocytes.

1. High-quality iPSCs (no differentiated colonies) were cultured before induction. 2–3 x 105 cells/well were plated in 6-well plates coated with Geltrex® matrix.

2. Cells were cultured in a 37˚C, 5% CO2 incubator for 1 or 2 days to reach about 15% confluence.

3. Additional cells were cultured as an un-induced control.

1. Cells were cultured in a 37˚C, 5% CO2 incubator to reach confluence with medium change to fresh Neural Expansion Medium every other day.

2. Cells can be further passaged and expanded in this medium. For NSCs at P0–P4, 5 µM ROCK inhibitor Y27632 should be included in the medium after cell plating.

3. Protocol described in the user manual was followed for cryopreservation.

1. Passage 0 NSCs were harvested on day 7 using Accutase® detachment solution.

2. Cells were resuspended in Neural Expansion Medium with 5 µM ROCK inhibitor Y27632 as described in the PSC Neural Induction Medium user manual.

3. Cells were plated at a density of 0.5 x 105 cells/cm2 onto Geltrex® pre-coated plates and cultured in a 37˚C, 5% CO2 incubator.

1. Protocol described in the Gibco® PSC Neural Induction Medium user manual was followed. Medium changed to PSC Neural Induction Medium on day 0, and changed to fresh medium every day from day 3 to day 6.

2. Cells were observed and images taken every day.

3. Cells that appeared different than neural cells (such as epithelial cells) were removed if observed during day 3 to day 6.

iPSC cultureDay 0 Change to PSC Neural Induction Medium

Day 7Harvest NSCs and re-plate in Neural Expansion Medium

Day 8–14NSC expansion and cryopreservation

Characterization

NSC generation CULTUREENGINEERINGDIFFERENTIATIONCHARACTERIZATION

We differentiated three PD, one MSA, and two age-matched control iPSC lines into NSCs using a novel neural induction and expansion medium. Our workflow is summarized in Figure 1. The starting iPSCs were cultured under feeder-free conditions in Essential 8™ Medium or StemPro® hESC SFM on Geltrex®- or Vitronectin-coated plates. Cells were induced according to the protocol described in the Gibco® PSC Neural Induction Medium user manual.

Representative phase-contrast images of cells during the 7-day neural

induction are shown in Figure 2A. No significant change in morphology was observed during the first two or three days of induction. On day 4, various morphologies were observed in some iPSC lines. Some lines displayed spiky edges and densely packed cells in the middle of the colonies. Despite the morphology differences during the induction, the resulting NSCs showed homogenous morphology after harvesting and re-plating in Neural Expansion Medium (Neurobasal® Medium, Advanced™ DMEM/F-12, Gibco® Neural Induction Supplement)

with the addition of 5 µM ROCK inhibitor Y27632 (Figure 2B).

Expanded NSCs were characterized by a variety of methods as described on the following pages. The karyotype of each line was analyzed and found to be normal, and NSC markers were observed by immunocytochemistry, flow cytometry, and TaqMan® qPCR.


Instrument highlight—EVOS® XL Imaging System

Cells were monitored and imaged throughout the neural induction process using the EVOS® XL Imaging System, a transmitted light imaging system that combines a digital camera, LCD display, and USB device storage. NSCs showed homogenous morphology after harvesting and replating in Neural Expansion Medium.

Figure 2. NSC generation from iPSC lines. iPSCs were cultured in Essential 8™ Medium on Geltrex® matrix–coated plates. NSC generation was performed according to the Gibco® PSC Neural Induction Medium user manual. (A) Phase contrast images are shown for the MSA and PD-3 cell lines on days 0, 1, 4, 5, and 7 of induction. (B) Phase contrast images for NSCs (passage 2) for all six lines after replating in Neural Expansion Medium. All images were captured using the EVOS® XL Imaging System.

PD-3

MSA

Day 0A

B

Day 1 Day 4 Day 5 Day 7

MSA Ctrl-2 PD-3 PD-2 PD-1 Ctrl-1

Life Technologies | Parkinson’s cell models10

Cytogenetic analysis

Monitoring the stability of the chromosomes is important to confirm that the cells are still free of chromosomal aberrations. Cytogenetic analysis was performed on 20 G-banded metaphase cells derived from the expanded NSCs. The results showed that all six NSC lines were karyotypically normal. Representative results are shown in Figure 3.

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C

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Figure 3. Cytogenetic analysis of NSCs. NSCs (PD-1, passage 9 (A); Ctrl-1, passage 2 (B); PD-2, passage 4 (C); and Ctrl-2, passage 2 (D)) were cultured in Neural Expansion Medium on Geltrex® matrix–coated plates for 2 to 3 days. Cells were then harvested for karyotype analysis. All clones tested demonstrated a normal karyotype, with no clonal abnormality detected at the G-banded level of resolution.

CULTUREENGINEERINGDIFFERENTIATIONCHARACTERIZATION


Figure 4. Immunocytochemistry demonstrated that NSCs from MSA and PD-3 donors express NSC markers. NSCs (passage 3) were cultured in Neural Expansion Medium on Geltrex® matrix–coated chamber slides for 2 days. Cells were stained for Nestin (green), Sox2 (red), Sox1 (green), and Pax6 (red) using the Human Neural Stem Cell Immunocytochemistry Kit. Blue: DAPI nuclear DNA staining. Images were captured using the EVOS® FLoid® Cell Imaging Station.

Immunocytochemistry for NSC markers

To evaluate the cells for expression of known NSC markers, the NSCs were expanded on Geltrex® matrix–coated chamber slides and tested for expression of NSC markers by immunocytochemistry at passage 3. All 6 NSC lines stained positive for markers Nestin, Sox2, Sox1, and some level of Pax6. Representative images are shown in Figure 4. All NSCs stained negative for the pluripotency marker Oct4 (data not shown).

Life Technologies | Parkinson’s cell models11

MSA

PD-3


Instrument highlight—Attune® Acoustic Focusing Cytometer

The Attune® Acoustic Focusing Cytometer was used to evaluate the percentage of expanded cells that stained positive for known NSC markers. With acoustic focusing, sample rates can be increased by over 10 times compared to hydrodynamic focusing, so utilizing the Attune® cytometer for this study allowed for a smaller starting sample. All NSC lines exhibited positive expression where expected.

Immunostaining and flow cytometry analysis for NSC markers

For a quantitative measure of NSC formation using Neural Induction Medium, the expanded NSCs were stained for Nestin, Sox1, Sox2, and Pax6, using Nanog as a negative control, followed by flow cytometry analysis. All iPSC-derived NSC lines exhibited positive (>90%) expression for Nestin, Sox1, and Sox2 and partial positive (25–50%) expression for Pax6, and were negative for Nanog expression. All flow cytometry samples were run and analyzed using the Attune® Acoustic Focusing Cytometer. Representative flow cytometry results for the MSA NSCs are shown in Figure 5. Figure 5. Immunostaining and flow cytometry analysis demonstrated that NSCs generated from MSA iPSC

line express known NSC markers. NSCs (passage 3) were cultured in Neural Expansion Medium on Geltrex® matrix–coated plates for 3 days. Cells were harvested, fixed, permeabilized, and stained with isotype control antibodies or antibodies for Nestin, Sox1, Sox2, Pax6, or Nanog. For the Sox1 staining, cells were further stained with Alexa Fluor® 488 donkey anti-goat secondary antibody. Flow cytometry analysis was performed using the Attune® Acoustic Focusing Cytometer. With the exception of Sox1, percent positive cells for each marker were determined by comparing to isotype controls. Percent positive cells for Sox1 were determined by comparing to secondary antibody–only stained samples.

Isotype controls Nanog/Sox2

9% 91%

51% 49%

Nestin/Pax6

2nd Ab only

0.2%

Anti-Sox1 and 2nd Ab

92% Sox1: 92% Sox2: 91% Nanog: 0% Nestin: 100% Pax6: 51%



Cell line authentication

To confirm the identity of the derived NSCs in comparison to both their parental iPSCs and the iPSCs’ parental fibroblasts, genomic DNA was isolated from PD fibroblasts, their subsequent reprogrammed iPSCs, and their subsequent differentiated NSCs. The AuthentiFiler™ PCR Amplification Kit was used to confirm that the NSCs shared the same profiles for short tandem repeat (STR), or “genetic fingerprints”, with their parental iPSCs and their subsequent parental fibroblasts. The AuthentiFiler™ PCR Amplification Kit is an STR multiplex PCR assay that amplifies 9 unique STR loci and the amelogenin gender-determining marker in a single PCR amplification. Briefly, STR targets were amplified using AuthentiFiler™ reagents on the Veriti® 96-Well Thermal Cycler. Resulting amplicons

were separated via capillary electrophoresis using a 3130xl Genetic Analyzer with 3130 POP-4® polymer and sample comparison was performed using GeneMapper® ID-X Software. STR profiles from each NSC sample were identical to the corresponding parental fibroblast and iPSC samples. A representative result is shown in Table 1. This suggests no significant gene duplication or deletion events occurred in the target loci chromosomes, and additionally, no cross-contamination events occurred that introduced other non-target human cells into the cell culture. These results provide confidence and verification that the NSC material is genetically identical to the starting fibroblast material.

Locus Chromosome PD-1 Fibroblasts PD-1 iPSCs PD-1 NSCs

D10S1248 10q26.3 14, 14 14, 14 14, 14

D1S1656 1q42.2 15, 16.3 15, 16.3 15, 16.3

AMEL X X, X X, X X, X

D2S1338 2q35 22, 26 22, 26 22, 26

D22S1045 22q12.3 16, 16 16, 16 16, 16

D19S433 19q12 13, 14 13, 14 13, 14

TH01 11p15.5 9, 9.3 9, 9.3 9, 9.3

D2S441 2p14 11, 11 11, 11 11, 11

D6S1043 6q16.1 12, 19 12, 19 12, 19

D12S391 12p13.2 17, 20 17, 20 17, 20

Table 1. Cell line authentication. Genomic DNA was isolated from PD-1 fibroblasts and their respective reprogrammed iPSCs and NSCs. The AuthentiFiler™ PCR Amplification Kit was used to confirm that NSCs shared the same short tandem repeat profiles (“genetic fingerprints”) with their parental fibroblasts and iPSCs. The numbers displayed in the table refer to the number of repeats on each allele.


Gene expression profiling of NSCs

To demonstrate that the generated NSCs express a broader panel of NSC marker genes, RNA was isolated from all 6 reprogrammed iPSCs and NSCs. After cDNA synthesis, gene expression levels were measured using the QuantStudio™ 12K Flex Real-Time PCR System and TaqMan® OpenArray® Human Stem Cell Panel, which contains 609 well-defined genes, plus 22 endogenous control genes. Gene expression profiling showed that NSC markers such as NESTIN, NCAM1, MESM1, NEUROD1, and NEUROG1

were up-regulated in the NSCs compared to those in their parental iPSCs, whereas pluripotency-specific genes such as OCT4, NANOG, NODAL, and REX1 were downregulated in the NSCs (Figure 6A). Hierarchical cluster analysis clearly distinguished the NSCs from their parental iPSCs and demonstrated that the NSCs derived in this study shared similar expression patterns with NSCs derived from iPSCs from BJ fibroblasts and H9 ESCs (Figure 6B).

Expression Level

A B

*

Figure 6. Gene expression profiles clearly distinguish NSCs from iPSCs. iPSCs were cultured in iPSC medium (KnockOut™ DMEM/F12, 20% KnockOut™ Serum Replacement (KSR), 10 mM non-essential amino acids, 2 mM GlutaMAX™-1, and 10 ng/mL bFGF) on MEFs for 4 days. NSCs (passage 3) were cultured in Neural Expansion Medium on Geltrex® matrix–coated plates for 3 days. Cells were harvested using TRI Reagent® before RNA isolation using RiboPure™ Kit and cDNA synthesis using SuperScript® VILO™ Master Mix. Expression levels were quantified using the QuantStudio™ 12K Flex Real-Time PCR System and TaqMan® OpenArray® Human Stem Cell Panel. (A) Expression levels of selected markers are shown relative to parental iPSCs. (B) A heat map shows distinct expression patterns of NSCs compared to their parental iPSCs and fibroblasts. The expression patterns of the NSCs are similar to other NSCs derived from iPSCs from BJ fibroblasts and H9 ESCs (denoted at the *).



Differentiation to dopaminergic neurons

To confirm that the derived NSCs have the potential to become dopaminergic (DA) neurons, NSCs from the Ctrl-2 and PD-3 lines were differentiated according to the method of Swistowski, et al. [5]. NSCs were first treated with DA induction medium containing Sonic Hedgehog and FGF8 for 10 days, followed by maturation in the presence of BDNF and GDNF. Immunocytochemistry was used to evaluate the presence of tyrosine hydroxylase (TH), a DA neuron marker and the rate-limiting enzyme in dopamine synthesis, FoxA2, a marker for floor plate–derived midbrain neurons, the neuronal marker β-III tubulin, and GIRK2 (G protein–activated inward rectifier potassium channel 2), a mature A9 subtype neuron marker. Representative images are shown in Figure 7.

A B C D E

F G H I J

Ctrl-2

PD-3

Figure 7. Immunocytochemistry demonstrated that NSC-derived DA neurons express DA and midbrain markers after 48 days of differentiation. NSCs (passage 4) cultured in Neural Expansion Medium were transferred to plates coated with poly-L-ornithine (20 µg/mL) and laminin (10 µg/mL). Cells were induced with DA Induction Medium (Neurobasal® Medium containing 1X B-27® Supplement, 100 µM MEM NEAA, 1X GlutaMax™-1 CTS™, 200 ng/mL Sonic Hedgehog, and 100 ng/mL FGF8) for 10 days followed by DA Maturation Medium (Neurobasal® Medium containing 1X B-27® Supplement, 100 µM MEM NEAA, 1X GlutaMax™-1 CTS™, 20 ng/mL BDNF, 20 ng/mL GDNF, and 200 mM ascorbic acid) for another 35 to 45 days. Phase-contrast images of Ctrl-2 (A) and PD-3 (F) on day 48. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton® X-100, and stained for: TH (green, B and G), TH (green) and FoxA2 (red) (C and H), TH (green) and β-III tubulin (red) (D and I), and GIRK2 (green, E and J). Blue: Hoechst® 33342 staining of nucleus. Images were taken using the EVOS® FLoid™ Cell Imaging Station.


Differentiation to glial cell types

To demonstrate that the derived NSCs have the capacity to differentiate into glial precursors and astrocytes, NSCs from the Ctrl-2 and PD-3 cell lines were induced with glial differentiation medium containing N-2 and B-27® Supplements for 22–30 days on Geltrex® matrix–coated plates. Immunocytochemistry was used to evaluate the presence of the glial precursor marker CD44 and the astrocyte marker GFAP. As shown in Figure 8, both control and PD donor–derived NSCs were able to differentiate into CD44-positive and GFAP-positive astrocytes.

Figure 8. Immunocytochemistry demonstrated that the derived NSCs have the capacity to differentiate into glial precursors and astrocytes. NSCs (passage 5) were plated on Geltrex® matrix–coated plates in StemPro® NSC SFM. Cells were induced with glial differentiation medium (DMEM/F-12 containing 1X N-2 Supplement and B-27® Supplement, 1X GlutaMax™-1 Supplement, and 1% FBS) for 22 to 30 days. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton® X-100, and stained for CD44 (red) and GFAP (green). (A) Ctrl-2 cell line, and (B) PD-3 cell line. Blue: Hoechst® 33342 staining of nucleus. Images were taken using the EVOS® FLoid® Cell Imaging Station.

A B

The EVOS® FLoid® Cell Imaging Station was used for analysis of the NSCs and differentiated NSCs in three different immunocytochemistry experiments. The three color channels plus phase channel allow for easy visualization of multiplexed staining and image merging.• The first experiment evaluated NSCs for the expression of neural markers, and all 6 NSC lines stained

positive for Nestin, Sox2, Sox1, and some level of Pax6.• The second experiment evaluated NSC-derived DA neurons for the expression of DA and midbrain markers

after 48 days of differentiation. These cells were found to express DA marker TH, midbrain marker FoxA2, and neuronal markers β-III tubulin and GIRK2.

• The third experiment evaluated NSC-derived glial precursors and astrocytes for the expression of glial markers after 22–30 days of differentiation. These cells were found to express glial precursor marker CD44 and astrocyte marker GFAP.

Instrument highlight—EVOS® FLoid® Cell Imaging Station


Gene expression profiling of differentiated NSCs

To further confirm the identity of cell types derived from these NSCs, total RNA was extracted from NSCs, DA neurons at various differentiation stages, and glial cells and astrocytes. Gene expression analysis was performed using the QuantStudio™ 12K Flex Real-Time PCR System and a custom TaqMan® OpenArray® Real-Time PCR Plate containing 45 genes known to be expressed in various neural cell types. Representative genes in the panel are shown in Table 2, and gene expression profiling data are shown in Figure 9.

Gene symbol Neural cell type Gene name Function

TH General neuron/DA neuron Tyrosine hydroxylase Oxidoreductase, oxygenase

EN1 Neural progenitor cell (NPC)/DA neuron Engrailed homeobox 1 Transcription factor, homeobox transcription factor

RALDH1 NPC/DA neuron Aldehyde dehydrogenase 1 family, member A1 Oxidoreductase, dehydrogenase

CALB1 DA neuron Calbindin 1 Select calcium binding protein, calmodulin-related protein

NR4A2 DA neuron Nuclear receptor subfamily 4, group A, member 2 Receptor, transcription factor, nuclear hormone receptor, nucleic acid binding

PITX3 DA neuron Paired-like homeodomain 3 Transcription factor, homeobox transcription factor, nucleic acid binding

DDC DA neuron DOPA decarboxylase (aromatic L-amino acid decarboxylase) Catalyzes the decarboxylation of L-3,4-dihydroxyphenylalanine (DOPA) to dopamine

RALDH2 NPC/DA neuron Aldehyde dehydrogenase 1 family, member A2 Oxidoreductase, dehydrogenase

EN2 NPC/DA neuron Engrailed homeobox 2 Transcription factor, homeobox transcription factor

MSX1 NPC/DA neuron Msh homeobox 1 Transcription factor, homeobox transcription factor, nucleic acid binding

FOXA2 NPC/DA neuron Forkhead box A2 Transcription factor, other transcription factor, nucleic acid binding

LMX1B NPC/DA neuron LIM homeobox transcription factor 1, beta Transcription factor, zinc finger transcription factor, other zinc finger transcription factor

NES NSC/General neuron Nestin Cytoskeletal protein, intermediate filament

SOX1 NSC SRY (sex determining region Y)-box 1 Transcription factor, HMG box transcription factor, nucleic acid binding

Table 2. List of genes showing differential expression between NSCs and DA differentiated cells on day 60.



Figure 9. Gene expression profiles of differentiated neurons or astrocytes. NSCs (passage 5 to 6) were differentiated toward dopaminergic (DA) neurons or glial cells/astrocytes. For DA neuron differentiation, NSCs cultured in Neural Expansion Medium were plated on plates coated with poly-L-ornithine (20 µg/mL) and laminin (10 µg/mL). Cells were induced with DA Induction Medium (Neurobasal® Medium containing 1X B-27® Supplement, 100 µM MEM NEAA, 1X GlutaMax™-1 CTS™, 200 ng/mL Sonic Hedgehog, and 100 ng/mL FGF8) for 10 days followed by DA Maturation Medium (Neurobasal® Medium containing 1X B-27® Supplement, 100 µM MEM NEAA, 1X GlutaMax™-1 CTS™, 200 ng/mL BDNF, 200 ng/mL GDNF, and 200 mM ascorbic acid) for another 40–50 days. For astrocyte induction, NSCs (passage 6) were plated on Geltrex® matrix–coated plates in StemPro® NSC SFM. Cells were induced with Induction Medium (DMEM/F-12 containing 1X N-2 Supplement and B-27® Supplement, 1X GlutaMax™-1 Supplement, and 1% FBS) for 22 to 30 days. Cells were harvested using TRI Reagent® before RNA isolation using RiboPure™ Kit and cDNA synthesis using SuperScript® VILO™ Master Mix. Expression levels were quantified using the QuantStudio™ 12K Flex Real-Time PCR System and a custom TaqMan® OpenArray® Real-Time PCR Plate. (A) Expression levels of markers in DA neuron samples are shown relative to starting NSCs. (B) Expression levels of GFAP in astrocyte samples are shown relative to starting NSCs. (C) Heat maps of NSCs, early DA neuron–differentiated cells, late DA neuron–differentiated cells, and astrocytes show distinct expression patterns.

Relative quantitation of DA neurons vs. NSCs

TH EN1 RALDH1 CALB1 NR4A2 PITX3 DDC RALDH2 EN2 MSX1 FOXA2 LMX1B NES SOX1-3

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Relative quantitation of GFAP expression inastrocyte-differentiated cells to starting NSCs

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Relative quantitation of DA neurons vs. NSCs

TH EN1 RALDH1 CALB1 NR4A2 PITX3 DDC RALDH2 EN2 MSX1 FOXA2 LMX1B NES SOX1-3

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Instrument highlight—QuantStudio™ 12K Flex Real-Time PCR System

The QuantStudio™ 12K Flex Real-Time PCR System was used for gene expression analysis of the NSCs and differentiated NSCs in two different characterization experiments:• The first experiment was a broad-based gene expression analysis of the NSCs with 609

genes in the TaqMan® OpenArray® Human Stem Cell Panel. NSC genes were shown to be up-regulated in all NSC lines, and NSCs were easily distinguished from parental iPSCs after results were plotted in a heat map.

• The second experiment was a broad-based gene expression analysis using a TaqMan® OpenArray® Real-Time PCR Plate containing 45 genes known to be expressed in various neural cell types. NSCs, early DA neuron–differentiated cells, late DA neuron–differentiated cells, and astrocytes were easily distinguished after results were plotted in a heat map.


C

Expression Level


Neural cells are characterized by high metabolic activity, which depends on the integrity of the mitochondria. Damaged mitochondria can lead to an overproduction of free radicals that harm cells and lead to apoptosis. Therefore, we utilized the PrestoBlue® Cell Viability Reagent to assess total metabolic activity within cells, and the MitoSOX™ Red Mitochondrial Superoxide Indicator along with the CellROX® Green Reagent to measure levels of destructive reactive oxygen species, such as superoxide, within mitochondria and whole cells. In addition, the Mitochondrial Membrane Potential Indicator (m-MPI) assay was used to determine whether mitochondrial membranes were still intact and able to carry the high membrane potential normally carried by healthy mitochondria. The CellEvent® Caspase-3/7 Green Detection Reagent was used to measure activation of caspases, which play a central role in the execution phase of cell apoptosis.

To evaluate the effects of a variety of cell stressors on NSCs, these assays were run in the presence of subsets of the following compounds:

• 6-OH-dopamine: neurotoxin that induces Parkinsonism in animals; kills neurons via reactive oxygen species (ROS)

• Rotenone: neurotoxin that inhibits the electron transport chain in mitochondria

• Valinomycin: potassium ionophore that disrupts the transmembrane concentration gradient

• TBHP (tert-butyl hydroperoxide): organic peroxide that induces oxidative stress

• CCCP: H+ ionophore that uncouples oxidative phosphorylation in mitochondria

• Staurosporine: general kinase inhibitor that induces apoptosis• PP242: mTOR inhibitor that activates autophagy • Chloroquine: Inhibits lysosome function; prevents

autophagosome turnover

Functional assays to assess NSC healthCell health is an important parameter to monitor when assessing the effect of drugs, environmental factors, and biological modifiers on cells. However, because cell health is not easily defined as a single physiological attribute, it is often desirable to use several different indices when determining cell health. To this end, the derived NSCs were expanded and tested with a panel of 5 assays that are particularly useful indicators of neural cell health, as each measures a different aspect of cell vitality (Table 3).

Assay Parameter measured Fluorescent readout

PrestoBlue® Cell Viability Reagent Metabolic activity Increased red fluorescence with higher metabolism

CellEvent® Caspase-3/7 Green Detection Reagent

Caspase-3/7–dependent apoptosis Increased green fluorescence in apoptotic cells

CellROX® Green Reagent Cellular oxidative stress Increased green fluorescence with increased cellular reactive oxygen species

MitoSOX™ Red Mitochondrial Superoxide Indicator

Mitochondrial superoxide levels Increased red fluorescence with increased mitochondrial superoxide levels

Mitochondrial Membrane Potential Indicator Mitochondrial membrane potential

Increased green/red fluorescence ratio with decreased mitochondrial membrane potential

Table 3. Selected assays that can be used to measure different aspects of neural cell health.


Figure 10. Protocol snapshots for a panel of functional assays used to assess the health of NSCs. The derived NSCs were expanded and utilized in functional assays that followed the experimental steps outlined here. Anticipated results in response to the addition of increasing concentrations of compounds that lead to cell stress are shown underneath each panel. Fluorescence images were taken with a Zeiss Axiovert® 25 microscope.

Toxicant

Toxicant

Toxicant

Toxicant* For TBHP, the compound was added 1 hr prior to adding PrestoBlue® reagent.

CellEvent® Capase-3/7Green Reagent

(Apoptosis)

Plate cells

Treat cells with compoundof interest (24 h at 37˚C)

Add a final conc. of 8 µM CellEvent® reagent to

the cells

Incubate for 30 min at 37˚C

Measure greenfluorescence

CellROX® Green and MitoSOX™ Red assay, multiplexed

(Reactive oxygen species)

Plate cells and incubate24 h at 37˚C

Add a final conc. of 5 µMMitoSOX™ and 7.5 µM CellROX®

to the cells (60 min at 37˚C)

Wash cells once in growth medium

Treat cells with compoundof interest for 2 h

Measure green and redfluorescence

PrestoBlue®

Cell Viability Reagent

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Plate cells

Treat cells with compoundof interest (24 h at 37˚C)*

Add 10X PrestoBlue® reagent to the cells

Incubate for 30 min at 37˚C

Measure red fluorescence

m-MPI Reagent

(Mitrochodral membranepotential)

Plate cells and incubate24 h at 37˚C

Add 2X m-MPI dye(30 min at 37˚C)

Treat cells withcompound of interest

Incubate 30 min at 37˚C

Measure green and redfluorescence

Protocol snapshotsBrief protocol snapshots are shown in Figure 10, and representative results for each assay are shown in Figure 11.

22 Life Technologies | Parkinson’s cell models22

Figure 11. A panel of functional assays was used to assess the health of the NSCs in response to various cell stressors. The derived NSCs were expanded on CELLstart™ CTS™ substrate in Neural Expansion Medium for 7 passages followed by StemPro® NSC SFM for another 4 passages. The NSCs were harvested and plated in CELLstart™-coated 384-well assay plates. The protocol snapshots shown in Figure 10 outline the experimental steps for each assay. A Tecan Safire2™ reader was used to measure fluorescence. Representative results are shown for (A) the PrestoBlue® assay on Ctrl-2, demonstrating the expected loss in metabolic activity with an increase in the concentration of stressors added, (B) the CellEvent® Caspase-3/7 Green assay on MSA, demonstrating the expected increase in apoptosis with an increase in the concentration of stressors added, (C, D) the multiplexed CellROX® Green assay and MitoSOX™ Red assay on PD-3, demonstrating the expected increase in oxidative stress with an increase in the concentration of stressors added, and (E) the m-MPI assay on Ctrl-1, demonstrating the expected decrease in mitochondrial membrane potential (higher green/red fluorescence ratio) with an increase in the concentration of stressors added.

PrestoBlue® Reagent (metabolism) Ctrl-2

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Summary

We have demonstrated a rapid, robust, and efficient method for the generation of neural stem cells from PD donor–derived iPSCs or normal iPSCs in 7 days. These NSCs display normal karyotypes, express known neural markers, and can be further differentiated to other neural cell types, such as DA neurons and glial precursors and astrocytes. We also have shown how these NSCs can be

used in a panel of functional assays to assess cell health in response to a variety of cellular stressors.

This work has set the stage for a “disease in a dish” model of Parkinson’s disease using fibroblasts from PD-affected donors. To understand the influence of specific genes on PD-relevant phenotypes, we will create isogenic cells that differ at a single,

defined locus (such as a single mutation in the LRRK2 gene). Work towards creating these matched control lines is underway in our laboratories. When paired with the differentiation and assay protocols that we have described, these lines will constitute a powerful set of model systems that can be used to study multiple aspects of PD-relevant biology.

References

1. Olanow CW, Stern MB, Sethi K (2009) The scientific and clinical basis for the treatment of Parkinson disease. Neurology 72(21 Suppl 4):S1–136.

2. Yan YP, Shin S, Jha BS, Liu Q, Sheng J, Li FH, Zhan M, Davis J, Bharti K, Zeng ZM, Rao M, Malik N, Vemuri MC (2013) Efficient and rapid derivation of primitive neural stem cells and generation of brain subtype neurons from human pluripotent stem cells. Stem Cells Transl Med 2:862–870.

3. Burn DJ, Jaros E (2001) Multiple system atrophy: cellular and molecular pathology. Mol Pathol 54:419–426.

4. Efthymiou A, Shaltouki A, Steiner JP, Jha B, Heman-Ackah SM, Swistowski A, Zeng X, Rao MS, Malik N (2013) Functional screening assays with neurons generated from pluripotent stem cell–derived neural stem cells. J Biomol Screen, Epub ahead of print.

5. Swistowski A, Peng J, Han Y, Swistowska AM, Rao MS, Zeng X (2009) Xeno-free defined conditions for culture of human embryonic stem cells, neural stem cells and dopaminergic neurons derived from them. PLoS One 4:e6233.

Ordering information

NSC culture and DA differentiation Quantity Supplier Cat. No.

Geltrex® LDEV-free hESC-qualified Reduced Growth Factor Basement Membrane Matrix 1 mL5 mL Life Technologies A1413301

A1413302

Gibco® PSC Neural Induction Medium 500 mL Life Technologies A1647801

Neurobasal® Medium 500 mL Life Technologies 21103049

Advanced™ DMEM/F-12 500 mL Life Technologies 12634010

MEM Non-Essential Amino Acids Solution (100X) 100 mL Life Technologies 11140050

B-27® Serum-Free Supplement (50X) 10 mL Life Technologies 17504044

GlutaMax™-1 CTS™ (100X) 100 mL Life Technologies A1286001

FGF-8b Recombinant Human Protein 100 µg Life Technologies PHG0271

Recombinant Human Sonic Hedgehog (SHH) 100 µg R&D Systems 1845-SH

StemPro® Accutase® Cell Dissociation Reagent 100 mL Life Technologies A1110501

GDNF Recombinant Human Protein 10 µg Life Technologies PHC7045

BDNF Recombinant Human Protein 10 µg Life Technologies PHC7074

Bovine Serum Albumin (BSA, 5%) 150 mg Life Technologies 15561020

L-Ascorbic acid 100 mg Sigma-Aldrich A4403

DPBS, no calcium, no magnesium 500 mL Life Technologies 14190144

Distilled water 500 mL Life Technologies 15230162

Natural Mouse Laminin 1 mg Life Technologies 23017015

Poly-L-ornithine hydrobromide 100 mg Sigma-Aldrich P3655

ROCK Inhibitor Y27632 5 mg EMD Millipore 688000

Neural Expansion Medium consists of Neurobasal® Medium, Advanced™ DMEM/F-12, and Gibco® Neural Induction Supplement. The recipe is found in the Gibco® PSC Neural Induction Medium user manual.


NSC culture and glial differentiation Quantity Supplier Cat. No.

Geltrex® LDEV-free hESC-qualified Reduced Growth Factor Basement Membrane Matrix 1 mL 5 mL Life Technologies A1413301

A1413302

StemPro® Neural Supplement 10 mL Life Technologies A1050801


DMEM/F-12, GlutaMAX™ 500 mL Life Technologies 10565018


FGF-Basic (AA 10-155) Recombinant Human Protein 50 µg Life Technologies PHG0026

EGF Recombinant Human Protein 10 µg Life Technologies PHG0314


N-2 Supplement (100X) 5 mL Life Technologies 17502048

B-27® Serum-Free Supplement (50X) 10 mL Life Technologies 17504044


Fetal Bovine Serum, Certified, US Origin 500 mL Life Technologies 16000044

StemPro® NSC SFM 1 kit Life Technologies A1050901

iPSC culture Quantity Supplier Cat. No.

Geltrex® LDEV-free hESC-qualified Reduced Growth Factor Basement Membrane Matrix 1 mL5 mL Life Technologies A1413301

A1413302

StemPro® hESC SFM 1 kit Life Technologies A1000701




Essential 8™ Medium 500 mL Life Technologies A1517001


Vitronectin (VTN-N) Recombinant Human Protein, Truncated 1 mL Life Technologies A14700

UltraPure™ 0.5 M EDTA, pH 8.0 4 x 100 mL Life Technologies 15575020

Collagenase, Type IV 1 g Life Technologies 17104019

2-Mercaptoethanol (55mM) 50 mL Life Technologies 21985023

FGF-Basic (AA 1-155), Recombinant Human Protein 10 µg Life Technologies PHG0264

Countess® Automated Cell Counter 1 instrument Life Technologies C10227

StemPro® EZPassage™ Disposable Stem Cell Passaging Tool 10 units Life Technologies 23181010

EVOS® XL Imaging System 1 instrument Life Technologies AME3300


Immunocytochemistry reagents Quantity Supplier Cat. No.Hoechst® 33342 10 mL Life Technologies H3570

Human Neural Stem Cell Immunocytochemistry Kit 1 kit Life Technologies A24354

GFAP (Glial Fibrillary Acid Protein) Rabbit Polyclonal Antibody 1 mL Life Technologies 180063

CD44 Rat Monoclonal Antibody 50 µg eBioscience 14-0441-81

GIRK2/KCNJ6 Goat Polyclonal Antibody 100 µg Thermo Fisher Scientific PA518802

Tyrosine Hydroxylase, Affinity Purified Rabbit Polyclonal Antibody 150 µL Pel-Freez® P40101

FoxA2 Mouse Monoclonal Antibody 100 µg Sigma-Aldrich WH0003170M1

β−III Tubulin Mouse Monoclonal Antibody 100 µg Sigma-Aldrich T8660

Alexa Fluor® 594 Goat Anti-Mouse IgG2b Antibody 0.25 mL Life Technologies A21145

Alexa Fluor® 594 Goat Anti-Mouse IgG (H+L) Antibody 0.5 mL Life Technologies A11005

EVOS® FLoid® Cell Imaging Station 1 instrument Life Technologies 4471136

10% Normal Goat Serum 100 mL Life Technologies 50062Z

1% Triton X-100 10 mL Life Technologies HFH10

Expression analysis Quantity Supplier Cat. No.RNAqueous®-4PCR Kit 30 isolations Life Technologies AM1914

SuperScript® VILO™ Master Mix 50 reactions Life Technologies 11755050

TaqMan® OpenArray® Human Stem Cell Panel 1 plate Life Technologies 4475390

TaqMan® OpenArray® Real-Time PCR Plate (custom) 1 plate Life Technologies Custom

QuantStudio™ 12K Flex Real-Time PCR System 1 system Life Technologies 4471090

TRI Reagent® 100 mL Life Technologies AM9738

RiboPure™ Kit 50 purifications Life Technologies AM1924

RNAlater® Stabilization Solution 100 mL Life Technologies AM7020

Attune® Acoustic Focusing Cytometer 1 system Life Technologies 4469120

Cell line authentication Quantity Supplier Cat. No.AuthentiFiler™ PCR Amplification Kit 50 tests Life Technologies 4479566

3130 POP-4® Polymer 7 mL Life Technologies 4352755

3130xl Genetic Analyzer (factory refurbished) 1 instrument Life Technologies 3130XL-R

Veriti® 96-Well Thermal Cycler 1 instrument Life Technologies 4375786


Functional assay reagents Quantity Supplier Cat. No. CELLstart™ CTS™ Substrate 2 mL Life Technologies A1014201

DPBS with calcium, magnesium 500 mL Life Technologies 14040133

Advanced DMEM/F-12 500 mL Life Technologies 12634010

Gibco® PSC Neural Induction Medium 500 mL Life Technologies A1647801

KnockOut™ DMEM/F-12 500 mL Life Technologies 12660012

FGF-Basic (AA 10-155) Recombinant Human Protein 10 µg Life Technologies PHG0024

EGF Recombinant Human Protein 10 µg Life Technologies PHG0314

StemPro® Neural Supplement 10 mL Life Technologies A1050801




PrestoBlue® Cell Viability Reagent 25 mL Life Technologies A13261

CellEvent™ Caspase-3/7 Green Detection Reagent 100 µL Life Technologies C10423

CellROX® Green Reagent 5 x 50 µL Life Technologies C10444

MitoSOX™ Red Mitochondrial Superoxide Indicator 10 x 50 µg Life Technologies M36008

Mitochondrial Membrane Potential Indicator 100 µL Codex Biosolutions CB80600-010

384-Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates 20/bag Corning 3712

Cell stressors Quantity Supplier Cat. No.6-OH-Dopamine 50 mg Tocris Bioscience 2547

Rotenone 250 mg Sigma-Aldrich 45656

Valinomycin 5 mL Sigma-Aldrich V3639

TBHP (tert-butyl hydroperoxide) 100 mL Sigma-Aldrich 458139

CCCP 500 mg Tocris Bioscience 0452

Staurosporine 100 µg Life Technologies PHZ1271

PP242 5 mg EMD Millipore 475988

Chloroquine 1g Sigma-Aldrich PHR1258


Find out more at lifetechnologies.com/parkinsonsFor Research Use Only. Not for use in diagnostic procedures. © 2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. The Parkinson’s Institute and Clinical Center is a trademark of Parkinson’s Institute. Hoechst is a registered trademark of Hoechst GmbH. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. Triton is a registered trademark of Union Carbide Chemicals & Plastics Technology Corp. Essential 8 is a trademark of Cellular Dynamics International, Inc. Accutase is a registered trademark of Innovative Cell Technologies. TRI Reagent is a registered trademark of Molecular Research Center, Inc. B-27 is a registered trademark of Southern Illinois University. Axiovert is a trademark of Carl Zeiss. Safire is a trademark of Tecan Group Ltd. Männedorf, Switzerland. CO34426 0114

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About Life Technologies Life Technologies Corporation is a global biotechnology company that is committed to providing the most innovative products and services to leading customers in the fields of scientific research, genetic analysis, and applied sciences. With a presence in more than 180 countries, the company’s portfolio of 50,000 end-to-end solutions is secured by more than 5,000 patents and licenses that span the entire biological spectrum—scientific exploration, molecular diagnostics, 21st century forensics, regenerative medicine, and agricultural research. Life Technologies has approximately 10,000 employees and had sales of $3.8 billion in 2012.

About the Parkinson’s Institute and Clinical Center Two and a half decades ago, a dedicated group of Parkinson’s disease experts set out to create a better way to research and provide care for Parkinson’s. We would go beyond your standard doctor-patient relationship. Patients would become partners in developing their care plans, improving their quality of life, and discovering new strategies to confront and manage their disease. Compassionate care would work hand in hand with cutting-edge research to seek answers, develop new therapies and treatments, and ultimately work toward a cure. Our result was the Parkinson’s Institute and Clinical Center.

Along the way, we have built something that’s thoroughly unique. Our Institute is a place where people living with Parkinson’s can both pursue their care and play an important role in research that will undoubtedly continue to change the landscape of Parkinson’s science and medicine. We are America’s only independent non-profit organization combining world-class care, clinical research, and laboratory research for Parkinson’s disease under one roof. By virtue of our design, we are

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