www.pnas.org/cgi/doi/10.1073/pnas.

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Supplementary Information for

Trans-differentiation of human adult peripheral blood T cells into neurons

Koji Tanabe, Cheen Euong Ang, Soham Chanda, Victor Hipolito Olmos, Daniel Haag,

Douglas Levinson, Thomas C. Südhof, and Marius Wernig

Marius Wernig

Email: wernig@stanford.edu

This PDF file includes:

Figs. S1 to S4

References for SI reference citations

1720273115

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Supplementary Information

SI Materials and Methods

Small molecule treatment. The transduced cells were treated by inhibitors following

concentrations from day 5. The medium was changed every 7 days; Forskolin (3µM, Sigma

Aldrich), Dorsomorphin (2µM, Tocris), SB431542 (10µM, Tocris), IWP2 (2µM,

StemGent), DAPT (10µM, Tocris), Retinoic Acid (0.5µM, Sigma Aldrich), SU5402

(10µM, Sigma Aldrich), Y27632 (10µM, Stemgent), SP600125 (10µM, Tocris).

Cell culture. Human iPSCs were maintained as feeder-free cells in E8 medium on Matrigel

(BD Biosciences)-coated 6-well plates (1). Mouse glial cells were derived from wild-type

CD1 mice. Briefly, mouse pups were sacrificed at postnatal day 3, the whole brain was

dissected, and cerebellum as well as olfactory bulb was removed. The residual brain was

homogenized and digested with trypsin (0.25%, Life Technologies) and 0.5 mM EDTA for

10 min at 37°C. Cells were dissociated by harsh trituration to kill neurons, and plated onto

10 cm tissue culture dishes in DMEM/NEAA/sodium pyruvate (Life technologies)

supplemented with 10% FBS. Upon reaching confluence, glial cells were trypsinized and

replated at lower density a total of two times to remove potential trace amounts of mouse

neurons before the glial cultures were used for co-culture experiments with iN cells. For

iN/glial cell co-cultures, glial cells were dissociated using trypsin/EDTA and seeded on

Matrigel-coated cover slips in 24-well plates two days before re-plating of iN cells. All

experiments were performed with APLAC and SCRO approval according to Stanford

University’s policies.

iN cell induction from sorted T cell population. The purified PBMC by Ficoll-density

gradient centrifugation (GE Healthcare) were stained with APC conjugated CD3 (Miltenyi

Biotec) and PE conjugated CD4 (Miltenyi Biotec) according to the manufacturer’s

instructions. Each population were sorted from PBMCs by FACS AriaII (Becton,

Dickinson and Company). BAMN plus EGFP were electroporated into each population

under the condition which was mentioned above.

Immunocytochemistry. Cells were fixed in 4% paraformaldehyde in PBS for 15 min at

room temperature. After fixation, cells were incubated in 0.1% Triton X-100 and 5%

Cosmic calf serum (CCS, Thermo Scientific) in PBS for 30 min at room temperature.

Primary and secondary antibodies were applied for 1 hour. The following antibodies were

used for our analysis: mouse anti-MAP2 (Sigma Aldrich, 1:500) and rabbit anti-Tuj1

(Covance, 1: 1,000). Alexa-488-, Alexa-546- and Alexa-633-conjugated secondary

antibodies (Life Technologies, 1: 1,000), 4’,6-Diamidino-2-phenylindole (DAPI) (Life

Technologies, 1:10,000) were used. For subtype marker staining, paraformaldehyde fixed

cells were incubated with a primary antibody overnight at 4ºC followed by incubation with

a secondary antibody for two hours. Antibodies used for neuronal subtype determination

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were: rabbit anti-Cux1 (Santa Cruz Biotechnologies, 1:50), rabbit anti-T2A (Millipore,

1:1,000), rabbit anti-v5 (Life Technologies, 1:1,000), mouse anti-huNCAM (Abcam,

1:100), mouse anti-Reelin (Millipore, 1:500), rabbit anti-Ctip2 (Santa Cruz

Biotechnologies, 1:500), mouse anti-Satb2 (Abcam, 1:200), rabbit anti-vGlut (Synaptic

Systems, 1:1,000), rabbit anti-vGAT (Synaptic Systems, 1:1,000), sheep anti-tyrosine

hydoxylase (PelFreeze, 1:750), mouse anti-human Nuclei (Millipore, 1:1,000), mouse anti-

peripherin (Millipore, 1:1,000), rabbit Ki67 (Abcam, 1:1000), goat Sox1 (R&D, 1:100) and

Gad2 (Developmental Studies Hybridoma Bank, 1:100). All images were taken with a

Zeiss epifluorescence microscope (Q-imaging) and three fluorescence channels were

combined using Adobe Photoshop.

Virus Generation. Lentiviruses were produced in HEK293T cells (ATCC, VA) by

cotransfection of lentiviral vector (12.5 µg) with the three packaging plasmids pRSV-REV

(3.125µg), pMDLg/pRRE (6.25 µg), and vesicular stomatitis virus G protein expression

vector (pVSVG, 3.125 µg) in a 10 cm tissue culture dish coated with poly-L-ornithine (15

µg/ml, Sigma Aldrich, MO). For transfection, the plasmid DNA mix and 75 µl linear

polyethylenimine (PEI, 1 µg/µl, Polyscience Inc., PA) was diluted separately in 500 µl

DMEM. Subsequently, both dilutions were vigorously mixed, incubated at room

temperature for 15 minutes, and added dropwise to the cells cultured in

DMEM/NEAA/sodium pyruvate (Life Technologies, MA) supplemented with 10% CCS

(GE Healthcare, UT). After 6-8 hours the complete media was changed and the virus

particles were harvested from the supernatant at 24 hours and 48 hours after infection. To

concentrate the virus, the supernatant was subjected to ultracentrifugation (23,500x g for 2

hours) and the pellet was suspended in DMEM with 0.5M sucrose yielding a 100-fold

enrichment. Concentrated virus was then aliquoted and snap frozen in liquid nitrogen. Only

virus preparation bathes with > 90% infection efficiency as assessed by EGFP expression

or puromycin resistance was used for subsequent experiments.

Generation of human iPS cell-derived neurons and co-culture with blood-iN cells.

Human iPSCs, which were established by episomal gene delivery, were dissociated using

Accutase (Innovative Cell Technologies) and plated as single cells in E8 medium

supplemented with Thiazovivin (2µM, Bio Vision) on 6-well plates coated with Matrigel

(BD Biosciences) to a final density of density of 3x105 cells/well (2). On the following day,

plated cells were infected with 5µl of concentrated virus of each Ngn2-Puro and rtTA

diluted in 1 ml E8 medium supplemented with Thiazovivin (2µM) to replace half of the

media per well. On day 0, half of the culture medium was replaced with N2 medium

(DMEM/F12 + N2 supplement (Life Technologies)) supplemented with insulin (10 µg/ml,

Life Technologies) and doxycycline (2 mg/l, Sigma Aldrich) to induce TetO gene

expression. On day 1, 2 µg/ml puromycin (Sigma Aldrich) was added and selection was

carried out for 48 hours with intermediate media changes. On day 4, ES-iN cells were

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washed with 0.5 mM EDTA in PBS and treated with Accutase. Dissociated cells were then

re-plated on blood-iN (14 days post-induction) cells co-cultured with glia in Neurobasal

medium supplemented with B27/Glutamax (Life Technologies) containing BDNF (10

ng/ml, PeproTech), NT3 (10 ng/ml, PreproTech), mouse laminin (200 ng, Life

Technologies), doxycycline (2 mg/l), and Ara-C (2 µM, Sigma Aldrich). After day 4, 50%

of the medium in each well was exchanged every 2-4 days. These blood iN cells were

induced without IL2 and dynabeads from day 3. The human iPSCs were ensured to be

mycoplasma-free by RT-PCR.

TCR recombination: Genomic DNA was harvested using DNA and Blood Kit (Qiagen).

The PCR reaction was performed using primer set B from TCRB Gene Clonality Assay

Kit (Invivoscribe) according the manufacturer’s protocol. The amplified products were

resolved using gel electrophoresis, TOPO-cloned into bacterial plasmids and sequenced to

confirm specificity of amplified products. The sequences were then uploaded to the IMGT

website (www.imgt.org/IMGT_vquest/vquest) to identify the V-D-J regions.

Electrophysiology. Electrophysiology experiments on blood-iN cells were essentially

performed as described before)(3). In brief, blood-iN cells only with elaborate

morphological complexity were patched using internal solution containing (for voltage

clamp, in mM) 135 CsCl2, 10 HEPES, 1 EGTA, 1 Na-GTP, and 1 QX-314 (pH 7.4, 310

mOsm) or (for current clamp, in mM) 130 KMeSO3, 10 NaCl, 10 HEPES, 2 MgCl2, 0.5

EGTA, 0.16 CaCl2, 4 Na2ATP, 0.4 NaGTP, and 14 Tris-creatine phosphate (pH 7.3, 310

mOsm), and extracellular solution containing (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2,

10 Glucose, and 10 HEPES-NaOH (pH 7.4). Current-clamp recordings for action potential

generation experiments and voltage-clamp recordings for AMPAR/GABAR-mediated

responses were performed at around −60 mV and −70 mV, respectively. Evoked responses

were generated using an extracellular concentric bipolar electrode (FHC) placed on the

culture monolayer at a distance of approximately 80 µm from the recording cell and

injecting 1-ms, 1-mA current through an Isolated Pulse Stimulator 2100 (A-M Systems)

connected to the stimulating electrode. The pharmacological agents were CNQX (25 µM,

Tocris), picrotoxin (50 µM, Tocris). The puff application of 50 µM AMPA (R-S AMPA

hydrobromide, Tocris) and GABA (γ-aminobutyric acid, Tocris) was performed for

100 ms using a Picospritzer III (Parker Instrumentation).

RNA-sequencing. Libraries produced were sequenced using the NextSeq mid-output

platform producing paired ends 2x75 reads. Raw reads were then mapped to the mouse

reference genome (mm9) using Tophat v1.3.0. RNA sequencing for PBMC and FACS

sorted EGFP+PSA-NCAM+ cells was done in biological duplicates. Expression levels for

the coding genes were estimated using the cuffdiff suite in Cufflinks v2.1.1. Candidates

genes which are >2 fold increased and had a P value of <0.05 (two-tailed) were filtered,

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clustered using GeneCluster 3.0 (4) and visualized as a heatmap using TreeView (5). The

gene ontology analyses were performed with Panther.

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Fig. S1: Optimizing the culture conditions to induce T cell-derived iN cells. (A)

Efficiencies of electroporation with BAMN and EGFP into PBMC as determined by EGFP

fluorescence after transfection at day 1. Each dot represents an individual donor (B)

Number of blood-iN cells on various feeder cells and coating conditions at day 21 from

1x106 PBMCs (N=3 individuals). (C) An example picture of blood-iN cells plated on glia

and human dermal fibroblasts (HDF) respectively. Scale bar = 50µm. (D) Graph showing

the relationship between induction efficiency vs electroporation efficiency for the 35

individuals without any inhibitors. (E) Pronounced glial cell death in T cell activation

conditions. Representative images of different, indicated T cell activation conditions show

strong glial cell death in +IL2; +Activator conditions but less in other conditions (white

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arrows). Scale bar 50 µm.

Fig. S2: Blood iN cells do not bypass a neural progenitor state. (A) Percentage of Ki67

positive cells among EGFP positive cells over the course of reprogramming (day 3 to 21)

n=3 individuals. (B) Representative immunofluorescence staining for Ki-67 and Sox1 at

day 3, 4, 5, 7, 9, 14 and 21 and hES-derived neural progenitor cells (positive control). Green

arrowheads represent EGFP+ and Ki67+ cells and red arrowheads represent EGFP+ Ki67-

cells). Scale bar: 50µm.

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Fig. S3: Stability of iN cells in the absence of sustained transgene expression. (A)

Immunofluorescence of iN cells with human specific NCAM (green), T2A (red) and DAPI

counterstain (blue) of blood-iN cells. Many human NCAM+ cells (white arrow heads) are

T2A negative (Scale bar: 50 µm). (B) BAMN and EGFP plasmids were transfected into

PBMCs. EGFP+ cells were purified 3 days later and plated on glia. FACS analysis on day

21 shows that among all human cells (defined as immunopositive for the human-specific

marker TRA 1-85) more than half of the PSA-NCAM+ iN cells had lost EGFP

fluorescence. (N=3 individuals). (C) RNA-sequencing results show that the exogenous

GFP, Ngn2-t2a-Ascl1, Brn2 and Myt1l decreased drastically in the GFP (-)/PSA-NCAM

(+) to the level of PBMCs compared to GFP (+)/PSA-NCAM (+).

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Fig. S4: PBMC-iN cells are predominantly derived from the CD3 positive T cell

population. (A) Frequency of the indicated four cell populations in freshly isolated

PBMCs based on flow cytometry. N=3 individuals. (B) Frequency of the four cell

populations among cells electroporated with EGFP. Note the strength transfection

preference for CD3+ cells. N=3 individuals. (C) Gel showing the TCRβ recombination

band at the predicted size range of 240-285bp for the 3 replicates of PBMC derived iN

cells. Red asterisks indicate unspecific bands. (D) Sanger sequencing of the three PCR

amplicons shown in (C) obtained after TOPO cloning confirmed VDJ rearrangement of the

TCRβ locus. V, D and J region are highlighted in red, blue and black respectively. (E)

Electroporation efficiency as determined by EGFP fluorescence in each of the 4 sorted cell

populations. N=3 individuals. (F) Passive intrinsic membrane properties of iN cells

derived from CD3+/CD4- (red) or CD3+/CD4+ (blue) T cells. (G) Sample traces of puff-

induced (red arrowheads) GABAA receptor- (left) and AMPA receptor- (right) mediated

currents and their corresponding peak amplitude and charge transfer from blood-iN cells

co-cultured with glia, demonstrating the presence of functional receptors. (H)

Quantification of GABA and AMPA puff experiments.

References

1. Beers J, et al. (2012) Passaging and colony expansion of human pluripotent stem

cells by enzyme-free dissociation in chemically defined culture conditions. Nat

Protoc 7(11):2029-2040.

2. Matsui T, et al. (2012) Neural stem cells directly differentiated from partially

reprogrammed fibroblasts rapidly acquire gliogenic competency. Stem Cells

30(6):1109-1119.

3. Chanda S, et al. (2014) Generation of induced neuronal cells by the single

reprogramming factor ASCL1. Stem cell reports 3(2):282-296.

4. Eisen MB, Spellman PT, Brown PO, & Botstein D (1998) Cluster analysis and

display of genome-wide expression patterns. Proc Natl Acad Sci U S A

95(25):14863-14868.

5. Saldanha AJ (2004) Java Treeview--extensible visualization of microarray data.

Bioinformatics 20(17):3246-3248.