Transdifferentiation of human adult peripheral bloodT cells into neuronsKoji Tanabea,b,1, Cheen Euong Anga,b,c,1, Soham Chandaa,b,d, Victor Hipolito Olmosa,b, Daniel Haaga,b,Douglas F. Levinsone, Thomas C. Südhofd,2, and Marius Werniga,b,2

aDepartment of Pathology, Stanford University, Stanford, CA 94305; bInstitute for Stem Cell Biology and Regenerative Medicine, Stanford University,Stanford, CA 94305; cDepartment of Bioengineering, Stanford University, Stanford, CA 94305; dDepartment of Molecular and Cellular Physiology andHoward Hughes Medical Institute, Stanford University, Stanford, CA 94305; and eDepartment of Psychiatry and Behavioral Sciences, Stanford University,Stanford, CA 94305

Contributed by Thomas C. Südhof, May 3, 2018 (sent for review November 21, 2017; reviewed by Thomas Graf and Hideyuki Okano)

Human cell models for disease based on induced pluripotent stem(iPS) cells have proven to be powerful new assets for investigatingdisease mechanisms. New insights have been obtained studyingsingle mutations using isogenic controls generated by genetargeting. Modeling complex, multigenetic traits using patient-derived iPS cells is much more challenging due to line-to-linevariability and technical limitations of scaling to dozens or morepatients. Induced neuronal (iN) cells reprogrammed directly fromdermal fibroblasts or urinary epithelia could be obtained frommany donors, but such donor cells are heterogeneous, showinterindividual variability, and must be extensively expanded,which can introduce random mutations. Moreover, derivation ofdermal fibroblasts requires invasive biopsies. Here we show thathuman adult peripheral blood mononuclear cells, as well asdefined purified T lymphocytes, can be directly converted intofully functional iN cells, demonstrating that terminally differen-tiated human cells can be efficiently transdifferentiated into adistantly related lineage. T cell-derived iN cells, generated by non-integrating gene delivery, showed stereotypical neuronal morphol-ogies and expressed multiple pan-neuronal markers, fired actionpotentials, and were able to form functional synapses. These cellswere stable in the absence of exogenous reprogramming factors.Small molecule addition and optimized culture systems haveyielded conversion efficiencies of up to 6.2%, resulting in thegeneration of >50,000 iN cells from 1 mL of peripheral blood in asingle step without the need for initial expansion. Thus, ourmethod allows the generation of sufficient neurons for experi-mental interrogation from a defined, homogeneous, and readilyaccessible donor cell population.

induced neuronal cells | direct conversion | transdifferentiation |disease modeling | iN cells

Advances in cell reprogramming and genome editing toolshave provided new ways to interrogate human gene function

in various human cellular contexts, such as neurons. In particu-lar, genetic engineering of embryonic or induced pluripotentstem (iPS) cells has proven powerful for dissecting the specificconsequences of disease-associated mutations in controlled ge-netic backgrounds (1, 2). However, these methods cannot beexpected to provide fully adequate cellular models of diseases forwhich highly polygenic mechanisms underlie risk. For example,large-scale genome-wide association study data suggest that 30–50% of the genetic risk for each of the neuropsychiatric disordersthat have been studied to date can be explained by the jointeffects of thousands of common genetic variants with small in-dividual effects, such that individual patients are likely to becarrying a unique combination of many contributory variants (3).One way to study such complex genetic backgrounds in human

neurons is by reprogramming patient cells to iPS cells (4).However, iPS cells have significant line-to-line variability interms of differentiation capacity, presumably due to variations intheir epigenetic and pluripotent state (5–7). Moreover, iPS cellsare often karyotypically unstable when grown in feeder-free

conditions, and their growth and formation is labor-intensiveand difficult to scale from a large number of individuals.Another way to obtain neurons is by deriving induced neuro-

nal (iN) cells from fibroblasts in a single conversion step, whichin principle would greatly facilitate their derivation from manypatients (8). However, unlike neonatal human fibroblasts, adulthuman fibroblasts have proven difficult to reprogram into syn-aptically competent iN cells (9–14). Moreover, fibroblasts areheterogeneous and ill-defined and must be expanded in vitrofrom invasive and painful skin biopsies to obtain sufficientnumbers, increasing the risk of acquiring random genetic muta-tions during an extended culture period. Here we report thatfunctional synapse-forming human iN cells can be induced fromfreshly isolated and stored adult peripheral T cells using non-integrating episomal vectors. Previous studies have shown theconversion of blood and urinary cells into various neural pro-genitor cells that only inefficiently gave rise to functional neu-rons (15–21). The described conversions were accomplished withtransient expression of iPS cell reprogramming factors, an

Significance

Recent advances in genomics have revealed that many poly-genetic diseases are caused by complex combinations of manycommon variants with individually small effects. Thus, buildinginformative disease models requires the interrogation of manypatient-derived genetic backgrounds in a disease-relevant celltype. Current approaches to obtaining human neurons are noteasy to scale to many patients. Here we describe a facile, one-step conversion of human adult peripheral blood T cells directlyinto functional neurons using episomal vectors without theneed for previous in vitro expansion. This approach is moreamenable than induced pluripotent stem cell-based approachesfor application to larger cohorts of individuals and will enablethe development of functional assays to study complex humanbrain diseases.

Author contributions: K.T., C.E.A., T.C.S., and M.W. designed research; K.T., C.E.A., S.C.,and V.H.O. performed research; D.H. and D.F.L. contributed new reagents/analytic tools;K.T., C.E.A., S.C., T.C.S., and M.W. analyzed data; and K.T., C.E.A., T.C.S., and M.W. wrotethe paper.

Reviewers: T.G., Center for Genomic Regulation; and H.O., Keio University Schoolof Medicine.

The authors declare no conflict of interest.

Published under the PNAS license.

Data deposition: The sequences reported in this paper have been deposited in the GeneExpression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no.GSE113804).1K.T. and C.E.A. contributed equally to this work.2To whom correspondence may be addressed. Email: tcs1@stanford.edu or wernig@stanford.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720273115/-/DCSupplemental.

Published online June 4, 2018.

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approach recently shown to induce a pluripotent intermediatestate (22).

ResultsDirect Induction of iN Cells from Peripheral Blood Mononuclear Cells.To investigate whether blood cells can be transdifferentiated toiN cells, we collected fresh blood from an adult healthy indi-vidual. We then isolated peripheral blood mononuclear cells(PBMCs) using gradient centrifugation and electroporated thesecells with episomal vectors encoding the four transcription fac-tors Brn2, Ascl1, Myt1l, and Ngn2, collectively termed theBAMN pool, which was previously found to generate iN cellsfrom human fibroblasts (9), and enhanced green fluorescentprotein (EGFP) into 3 million PBMCs. Transfected cells werethen cultured in IL-2 and CD3/CD28 antibodies containingmedia supporting T cell growth (Fig. 1A). On day 3, we placedthe nonadherent, transfected blood cells on different substrates,including primary mouse glia, mouse fibroblast SNL cell line,human primary fibroblasts, Matrigel, Polyornithine, or laminin-coated dishes. BAMN-transfected cells attached well on primary

mouse glia cells and human fibroblasts, but on no other sub-strate. Cells transfected with EGFP alone did not attach to anysubstrate (SI Appendix, Fig. S1 B and C). On day 5, we changedN3 medium

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Fig. 1. Generation of neuronal cells from peripheral blood cells. (A) Experi-mental outline of iN cell induction from PBMCs. (B) Morphological changesduring iN cell induction from PBMCs. (Scale bars: 50 μm.) (C) The relativenumber of iN cells from T cells with or without T cell activator (anti-CD3/CD28),with or without IL-2, or a change to N3 media on day 3. n = 3 individuals. (D)Efficiency of iN cell induction of transduced cells from 35 individual donorswithout inhibitors at day 21. n = 1 for each donor. The number of iN cells onday 21was divided by the number of total EGFP+ cells counted on day 1. (E andF) Relative iN cell induction (E) and efficiency of electroporation (F) fromPBMCs of three individual donors that were kept at −80 °C or at 4 °C for 2 drelative to the fresh sample. *P < 0.05, paired t test. (G) Transdifferentiationefficiency of PBMCs from three individual donors kept at −80 °C or at 4 °C for2 d relative to the fresh sample. Error bars represent SD.

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Fig. 2. Small molecule treatment improves iN cell conversion efficiency andmaturation. (A) Immunofluorescence analysis of iN cells with and withoutsmall molecules (3sm, three small molecules: forskolin, dorsomorphin, andSB431542; Cont, DMSO). (Scale bars: 50 μm.) (B) Fold change of improved iNcell formation on day 21 following various small molecule treatments as in-dicated. Do, dorsomorphin; Fo, forskolin; SB, SB431542. Data shown are av-erage fold changes of three independent experiments using PBMCs fromthree different donors. The fold change over the control condition was plottedbecause the absolute reprogramming efficiency was variable among the three do-nors, but the fold change was consistent. *P < 0.05, paired t test. The error barsindicate SDs. Similar results were obtained with PBMCs from another set of threedifferent donors. (C) Example traces of action potential firing recorded from PBMC-derived iN cells with or without 3sm at days 21 and 42 (n represents the number ofcells patched that shows action potentials over the total number of cells patched).The experiment was performed with cells from three different donors, yieldingsimilar results. (D) Sample traces (Left) and average values (mean ± SEM; Right)demonstrating the presence of voltage-gated Na+ and K+ channels in blood iN cellscocultured with glia with 3sm for 42 d. (Inset) Expanded view of the dotted boxedarea. (E) Intrinsic properties of membrane potential (Vrest), capacitance (Cm), andinput resistance (Rm) approach more mature values over time. (F) Maturation ofblood iN cells on extended coculture with glia in 3sm from day 21 to day 42 asdetermined by increased action potential (AP) height and threshold. Bar graphsrepresent mean ± SEM. *P < 0.05; **P < 0.01. ns, not significant.

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the hematopoietic medium to the neuronal medium N3. Re-markably, after seeding of the human blood cells on murine glialcells, we noticed that glial cells deteriorated quickly. We reasonedthat the nontransfected, activated human T cells presumably be-gan to attack the mouse glia. Withdrawal of IL-2 and the T cellactivators after day 3 not only mitigated this problem, but alsoimproved reprogramming by 2.7- to 3.6-fold. Switching to neuro-nal media on day 3 also rescued glial viability but improvedreprogramming only slightly (Fig. 1C and SI Appendix, Fig. S1E).To test the general applicability of our protocol, we obtained

blood from a total of 35 healthy adult donors of various ages andethnicities and both sexes. Surprisingly, the electroporationefficiencies varied significantly (SI Appendix, Fig. S1A), butnonetheless we were able to generate morphologically complexiN cells from all tested blood samples (Fig. 1D). The reprog-ramming efficiency varied as well, but the electroporation ratedid not correlate with the reprogramming efficiency (SI Appen-dix, Fig. S1D). Unlike iPS cell reprogramming, reprogrammingto iN cells was consistently lower in aged donors (Fig. 1D).Because most biorepositories freeze patient samples, we next

tried to induce iN cells from short- and long-term stored PBMCsat cold temperature. We isolated PBMCs from a fresh whole-blood sample, reprogrammed a fraction of the cells, and frozethe remaining cells at −80 °C. Another fraction of the whole-blood sample was maintained at 4 °C for 2 d before subsequentPBMC isolation. We then reprogrammed the PBMCs isolatedfrom the stored blood sample and the frozen PBMCs. Therewere no significant differences in the reprogramming efficiency andelectroporation efficiency between fresh and frozen PBMCs, but theiN cell yield was substantially lower from PBMCs stored at 4 °C dueto decreased transfection efficiency (Fig. 1 E–G). Thus, storage offreshly isolated cells at −80 °C did not affect reprogramming.

Combined BMP and TGF-β Pathway Inhibition and PKA ActivationImproved Reprogramming Efficiency. We next sought to furtherincrease the induction efficiency of iN cells using small mole-cules. Blockade of BMP and TGF-β pathways have been shownto promote neural induction during normal development, duringES cell differentiation, and from fibroblasts (23–25). Moreover,cAMP has been reported to facilitate neuronal survival andmaturation (26). Therefore, we treated reprogramming bloodcells with compounds regulating these three pathways from day5 to day 21 (Fig. 2A). Indeed, the number of iN cells was sig-nificantly increased by the adenylyl cyclase activator forskolin(1.9-fold), the BMP pathway blocker dorsomorphin (3.7-fold),and the TGF-β pathway inhibitor SB431542 (4.6-fold) whenanalyzed on day 21 (Fig. 2B). The combination of the three in-hibitors (forskolin, dorsomorphin, and SB431542; 3sm) showedan additive effect on the number of iN cells relative to the cellsseeded (8.7-fold) (Fig. 2 A and B). These combined improve-ments yielded a reprogramming efficiency of up to 6.2%, whichtranslates into 54,265 iN cells from 1 mL of blood (Fig. 2B).

Blood iN Cells Exhibit Passive and Active Neuronal MembraneProperties. We then tested the functional properties of bloodiN cells that were generated with and without addition of the threeinhibitors. Patch-clamp recordings of blood iN cells showed theirability to fire action potentials on step-current injection at 21 and42 d after infection and expressed functional voltage-gated Na+and K+ channels (Fig. 2 C and D). As expected, 3sm treatmentand longer culture periods (day 42) yielded cells with parametersof more mature action potentials (height, threshold, faster de-polarization and repolarization kinetics) and more mature in-trinsic properties, such as increased capacitance and decreasedinput resistance, compared with control-treated cells and day21 cells, respectively (Fig. 2 C, E, and F).

ROCK Inhibition Substantially Improves Formation of NeuronalMorphologies but Does Not Improve Functional Properties. In an at-tempt to increase the reprogramming efficiencies even further, wescreened six additional compounds—IWP2, DAPT, retinoic

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Fig. 3. ROCK inhibition improves morphological maturation, but notfunctional maturation. (A) Relative number of TUJ1-positive (yellow bars) orMAP2-positive (blue bars) cells with 3sm (forskolin, dorsomorphin, andSB431542) and additional small molecules from PBMCs from three individualdonors on day 21, normalized to the no treatment control condition (Cont).n = 3 individuals. *P < 0.05 relative to DMSO (only with 3sm). (B) Relativeaverage length of neurites with small molecules from PBMCs from threeindividual donors. The length was normalized to the no treatment control(Cont). n = 3 individuals. *P < 0.05 relative to the DMSO. (C) Example pic-tures of iN cells with indicated inhibitors. Green, EGFP fluorescence (Scalebars: 50 μm.). (D) iN cells generated with 3sm plus ROCK inhibitor expressneuronal markers, including MAP2. (Scale bars: 50 μm.) (E) The effectof indicated small molecule combinations on reprogramming efficiency.*P <0.05, paired t test. n = 3 individuals. (F) The effect of the duration of 3smplus ROCK inhibitor with (yellow bars) or without (blue bars) GDNF andBDNF on reprogramming efficiency. n = 3 individuals. (G) Representativetraces of action potential responses of PBMC-derived iN cells under controlcondition (N3 only; Top) and in the presence of 3sm plus ROCK inhibitorwhen cocultured with glia for 21 d (Left) or 42 d (Right). (H) Graph showingthe total number of neurons in four conditions: DMSO control (black line), 3smtreatment for 21 d (blue line), 3sm plus ROCK inhibitor for 21 d (red line), and3sm plus ROCK for 14 d and 3sm for 7 d. n = 3 individuals. *P < 0.05.

6472 | www.pnas.org/cgi/doi/10.1073/pnas.1720273115 Tanabe et al.

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acid, SU5402, Y26732, and SP600125—targeting pathways pre-viously implicated in neural differentiation in combination withforskolin, dorsomorphin, and SB431542. Of the single com-pounds tested, we found that ROCK inhibition increased boththe relative number of iN cells and the morphological com-plexity of reprogramming cells compared with the DMSOcontrol (Fig. 3 A–D). Other combinations of small mole-cules did not have additional effects (Fig. 3E). Moreover, theaddition of neurotrophic factors did not substantially increasethe formation of iN cells, but long-term drug treatmentyielded more iN cells than transient small molecule treatment(Fig. 3F).Importantly, however, when we tested their functional prop-

erties, cells generated in the presence of ROCK inhibition werenot able to generate mature action potentials, unlike cells grownwithout the ROCK inhibitor (Fig. 3G). The lack of mature actionpotentials suggests that ROCK inhibition simply affects cyto-skeletal rearrangements but perturbs functional neuronal matu-ration. To assess whether transient ROCK inhibition is sufficientto increase conversion efficiency and still allow for functionalmaturation, we removed the ROCK inhibitor and 2 wk after 3smplus ROCK inhibitor treatment and found no beneficial effect(Fig. 3H).

Molecular Characterization of Blood-Derived iN Cells. To assess thetranscriptional changes induced by reprogramming, we per-formed RNA-sequencing (RNA-seq) on the donor PBMCs aswell as on EGFP+ and PSA-NCAM+ blood iN cells purified bymagnetic cell sorting. A total of 6,941 genes were differentiallyexpressed between the PBMCs and blood iN cells (Fig. 4A). Theup-regulated genes were enriched for Gene Ontology terms suchas nervous system development and synaptic transmission, whilethe down-regulated genes were enriched for cellular defense

responses (Fig. 4A). Pan-neuronal markers (TAU, TUBB3,MAP2, and NCAM) were up-regulated while blood surfacemarkers (CD8, CD45, and CD3) were silenced (Fig. 4 B and C).As expected, proproliferative genes were down-regulated andnegative cell cycle regulators were induced (Fig. 4 D and E).These results suggest that blood iN cells have silenced hemato-poietic transcriptional programs and have adopted a pure neuronalidentity.To characterize the regional and neurotransmitter identity of

PBMC-derived iN cells, we performed immunofluorescenceand considered the results in conjunction with the RNA-seqdata. The two methods yielded very consistent results. Wefound that blood iN cells expressed the vesicular glutamatetransporter (VGLUT) but not the vesicular GABA transporter,GAD65, or tyrosine hydroxylase, suggesting that blood iN cellsare excitatory neurons similar to fibroblast iN cells and Ngn2-ES iN cells (8, 9, 27) (Fig. 4 F, G, and I). Based on the RNA-seqresults, we found expression of forebrain markers and corticallayers II–V but not layers I, IV, and VI (Fig. 4 H and J). Im-munofluorescence analysis demonstrated that in fact almost allblood iN cells were positive for SATB2 and CTIP2 but negativefor REELIN, as suggested by the RNA results (Fig. 4 F and G).SATB2/CTIP2 double-positive cells are found in layer V of themouse cortex (28).

The Blood-to-Neuron Conversion Does Not Involve a ProliferativeNeural Progenitor State. Several groups have reported the con-version of blood cells into proliferative neural cells using subsetsof iPS cell reprogramming factors (20, 21). In contrast, ourreprogramming factors are not proproliferative and our ap-proach yields postmitotic neurons directly. Nevertheless, it maybe possible that even our reprogramming process involves aproliferative intermediate progenitor. To address this question,

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Synaptic transmissionChromatin organizationMAPK cascadeNervous system developmentCellular component morphogenesisIntracellular protein transportSensory perception of smell

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Fig. 4. Blood iN cells express genes characteristic ofexcitatory, postmitotic neurons. (A) Heatmap show-ing 6,941 genes differentially expressed between PBMCand blood iN cells. Two biological replicates per pop-ulation, greater than twofold change and P < 0.05.Shown are the seven most significant (P < 0.05, Bon-ferroni-corrected) Gene Ontology terms among up- anddown-regulated genes using PANTHER. (B) Induction ofpan-neuronal markers. (C) Suppression of blood cellgenes. (D) Down-regulation of cell cycle activators. (E)Induction of antiproliferative cyclin-dependent kinaseinhibitors. (F) Immunofluorescence of blood iN cellsshowing expression of the excitatory marker vGLUTand subtype markers SATB2 and CTIP2 at 21 d afterinfection and cultured with 3sm on glia (Scale bars:50 μm.). (G) Quantification of day 21 blood iN cellsgrown on glia in 3sm conditions by immunofluores-cence for indicated markers. (H) Expression of region-specific markers by the PBMC-derived iN cells by RNA-seq. (I) Validation of the neurotransmitter-specificmarkers by RNA-seq. (J) Validation of the cortical sub-type-specific markers by RNA-seq.

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we decided to stain the cells at different time points betweendays 3 and 21 during reprogramming with the neural progenitormarker Sox1 and the proliferation marker Ki67 (SI Appendix,Fig. S2). To our surprise, Ki67-positive cells decreased only afterthe first week of reprogramming. Nonetheless, all Ki67-positivecells were Sox1-negative, and the number of proliferative cellsdeclined rapidly after day 7. Thus, no proper neural progenitorcells are formed as transient intermediates. The initial persis-tence of proliferative cells is likely due to the cytokines that weused to activate lymphocytes.

Blood iN Cells Are Stable Without Persistent Transgene Expression.To examine whether the neuronal identity is dependent oncontinued transgene expression, we first examined whetherthe transgenes were perhaps already silenced in our original

transfection protocol (Fig. 1A). We had used a bicistronic de-livery of Ngn2 and Ascl1 linked by the T2A self-cleaving peptide.After cleavage, the T2A peptide sequence is predicted to befused in frame with Ascl1 and thus can serve as a tag of theexogenous Ascl1. Immunostaining with T2A antibodies showedthat as many as 40% of blood iN cells were T2A-Ascl1 negative,and FACS analysis demonstrated that approximately the samefraction of cells had silenced the EGFP transgene, which was co-electroporated together with the reprogramming factors (SIAppendix, Fig. S3 A and B). Because we used the mouse cDNAsfor all four reprogramming factors to reprogram human cells, wecould accurately distinguish the exogenous (mouse) factors fromthe endogenous (human) ASCL1, NGN2, BRN2, and MYT1Lgenes in our RNA-seq dataset. This analysis clearly demon-strates that the exogenous factors were effectively silenced inthe EGFP−/PSA-NCAM+ iN cell population (SI Appendix,Fig. S3C). Thus, blood iN cells can adopt a stable neuronalidentity without the need for continued expression of exogenousreprogramming factors.

Synaptically Competent iN Cells Can Be Derived from CD3+ T Cells.PBMCs consist of fairly heterogenous hematopoietic cell pop-ulations. We wondered whether iN cells could be establishedfrom a more defined cell type. After characterizing freshlytransfected cells, we found that our electroporation conditionsgreatly favor CD3+ T cells, suggesting that the vast majority ofPBMC iN cells are in fact T cell-derived (SI Appendix, Fig. S4 Aand B). To more specifically test whether T cells can be con-verted, we introduced the BAMN factors into four defined cellpopulations purified based on CD3 and CD4 expression. Indeed,morphologically complex iN cells were induced from CD3+/CD4−and CD3+/CD4+ cells, but not from CD3− cells (Fig. 5A). T cell iNcells showed passive and active neuronal membrane propertieswhen cocultured with glia for 18 d and recorded on day 21 (Fig. 5B and C and SI Appendix, Fig. S4E). Finally, we confirmed theT cell origin of PSA-NCAM FACS-purified iN cells, by dem-onstrating genomic VDJ rearrangements at TCR-β locus (SIAppendix, Fig. S4 C and D).The defining property of a neuron is its ability to make syn-

aptic connections. Therefore, we asked whether BAMN-inducedT cell iN cells would be able to form functional synapses. Localadministration of GABA and AMPA on the soma and proximaldendrites of day 21 T cell iN cells recorded in voltage-clampmode yielded prominent GABAA receptor-mediated inhibitorypostsynaptic currents (PSCs) and AMPA receptor-mediated ex-citatory PSCs, respectively (SI Appendix, Fig. S4 G and H),demonstrating the presence of functional neurotransmitter re-ceptors. To address whether the T cell iN cells have the capacityto form synapses and functionally integrate into existing neuro-nal networks, we plated EGFP-labeled iN cells at 14 d post-induction onto unlabeled human iPS cell-derived neurons. At21 d after coculture, we performed voltage-clamp recordingsfrom EGFP-positive cells and observed synaptic AMPA receptor-mediated spontaneous network activities, indicating their success-ful integration into the synaptic network (Fig. 5D). In addition,spontaneous PSCs could also be observed in these cells, as con-firmed by application of the specific AMPA and GABAA receptorantagonists CNQX and picrotoxin, respectively (Fig. 5D). Finally,evoked PSCs could also be elicited by activating surrounding axonsvia extracellular field stimulation of the vicinity (Fig. 5E), unam-biguously demonstrating that blood iN cells can receive functionalsynaptic inputs from other neurons.

DiscussionOur demonstration that adult human peripheral T cells can bedirectly converted to neurons has both conceptual and practicalimplications. During normal development, the only cells with thepotential to change lineage identity are uncommitted stem andprogenitor cells. Most reprogramming studies use heterogeneousfibroblasts as donor cells, raising the question as to whether thetransdifferentiation capability is limited to undifferentiated progenitor

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Fig. 5. Generation of synaptically competent iN cells from T cells. (A) Rel-ative reprogramming efficiency of iN cells from the four indicated PBMCpopulations. The plotted efficiency was normalized by the electroporationefficiency and the efficiency of the CD3+/CD4− cell population was set to 1(n = 3 individuals). (B) Recording configuration of EGFP-labeled blood iN cellscocultured with unlabeled iPS cell-derived neurons. The recording electrode(Rec) was placed onto an EGFP-positive blood iN cell (white arrowhead)surrounded by nonfluorescent human iPS cell-derived neurons (black ar-rowheads). (Scale bar: 50 μm.) (C) Example traces of action potential firingrecorded from iN cells derived from CD3+/CD4− (red) or CD3+/CD4+ (blue)T cells. N represents the number of cells patched that show action potentialsover the total number of cells patched. (D) Representative traces of AMPAreceptor-mediated spontaneous network activity (Top, black) recorded froma T cell-derived iN cell, indicating successful integration into the humansynaptic network. The trace in red (Bottom) represents an expanded view ofthe boxed area. Spontaneous PSCs recorded from a T cell-derived iN cell(Top, black) and subsequently blocked by CNQX and picrotoxin (Bottom,black). The trace in red (Middle) represents and expanded view of the boxedarea. This pattern was observed in 2 of the 27 cells patched. (E) Evoked PSCs(five trials) in response to extracellular field stimulation recorded from ablood iN cell (Top), which was subsequently blocked by CNQX and picrotoxinapplication (Bottom).

6474 | www.pnas.org/cgi/doi/10.1073/pnas.1720273115 Tanabe et al.

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cells (29). Our results unequivocally show that terminally dif-ferentiated cells can be transdifferentiated into another, distantlyrelated somatic lineage.The derivation of neurons from adult peripheral blood cells

also has important practical implications. Unlike fibroblasts,whose derivation requires an invasive and painful skin punchbiopsy, lymphocytes can be obtained in large numbers from asimple venipuncture, a procedure performed in almost everyhospitalized patient, often on a daily basis. Moreover, bloodsamples are stored in biorepositories in much larger numbersthan skin fibroblasts. Of relevance for blood iN cell applicationsusing such repositories, we observed that iN cells can be obtainedfrom fresh and frozen blood cells with similar efficiency.Therefore, our blood iN cell conversion described here enablesthe generation of human neurons from virtually any individual,unlike the use of fibroblasts as donor cells, which have provendifficult to obtain from certain populations, such as children andmentally ill persons. In addition, the greater accessibility allowsfor scalability of donor individuals, which will be instrumental inassessing how common, low-risk–conferring genetic variantscontribute to cellular function in complex genetic diseases. An-other advantage over fibroblasts as donor cells is that fibroblastsneed to be expanded in vitro to obtain sufficient numbers, whichmay lead to accumulation of deleterious mutations.From a mechanistic standpoint, it was unexpected to find that—

unlike iN cell transdifferentiation from fibroblasts—the earlycoculture of glia was critical for transdifferentiation of bloodcells. The effect of glia seems to be fundamentally different herethan in fibroblast reprogramming, where glial coculture does notsubstantially impact conversion efficiency rather than synapticmaturation (8, 9). In contrast, the role of glial factors affectedthe generation of iN cells in general when transdifferentiatedfrom blood cells. Since transfected PBMCs also attached onto a layerof fibroblasts but did not reprogram, we assume that the monolayerof glial cells provides secreted or cell contact-dependent factorsto the blood cells that are essential for transdifferentiation inaddition to enabling their attachment.While this paper provides a clear proof of concept that human

adult peripheral T cells can be converted to iN cells with all keybiochemical and functional properties of neurons, we note that—similar to human fibroblast iN cells—these cells exhibit less maturesynaptic properties compared with primary mouse or iPS cell-

derived iN cells (20, 21, 30, 31), and using the current protocol,our blood iN cells are exclusively excitatory. While many cellbiological processes, such as transcription, polarization, migra-tion, and subcellular transport, can already be studied in thesecells, future efforts will need to focus on improving synapticmaturation and deriving additional neuronal subtypes.

Materials and MethodsPBMCs were isolated from fresh blood donations obtained through theStanford Blood Bank from individuals of various ethnic backgrounds (Cau-casian, Japanese, Indian, South American, and African), various ages (16–78 y), and both sexes using density gradient centrifugation with Ficoll-PaquePLUS (GE Healthcare) according to the manufacturer’s instructions. PBMCswere frozen by a stem cell banker (ZENOAQ). Then 3 μg of vectors (PcxLE-Ngn2-2A-Ascl1, Brn2, and Myt1L: 0.666 μg; pCXLE-GFP: 0.5 μg; pCXWB-Ebna1: 0.5 μg) or nonreplicative vectors (pCXWB-Brn2, Ascl1, v5Myt1l,flagNgn2 and GFP: 0.5 μg) were electroporated into 3 × 106 isolated PBMCswith the Nucleofector 2b Device (Lonza) with the Amaxa Human T-CellNucleofector Kit, program V-024 (Lonza).

Transduced cells were cultured for 3 d in six-well plates in X-VIVO10medium (Lonza) supplemented with 30 U/mL IL-2 (PeproTech) and 3.4 μL/mLDynabeads Human T-Activator CD3/CD28 (Life Technologies). At 3 d afterelectroporation, 0.1–1 × 106 transduced cells were seeded on primary gliaculture in a well of a 12-well plate. Glia (1.5 × 105) were seeded in a well of a12-well plate coated with Matrigel (Corning). At 2 d after seeding, the mediumwas replaced with DMEM/F12 (Invitrogen) including N2 supplement (Gibco),B27 supplement (Gibco), and insulin (5 μg/mL; Sigma Aldrich). The mediumwas changed every 7 d. The RNA-seq files are available in the NationalCenter for Biotechnology Information’s Gene Expression Omnibus database(accession no. GSE113804). Virus generation, electrophysiology, RNA-sequencing,TCR recombination and generation of human embryonic stem cell-derivedneurons are described in detail in SI Appendix.

ACKNOWLEDGMENTS. We thank Dr. Keisuke Okita, Dr. Kazutoshi Takahashi,and members of the M.W. laboratory for important suggestions. This projectwas supported by National Institutes of Health Grants R01 MH092931 andU19 MH104172, the New York Stem Cell Foundation (NYSCF)–RobertsonPrize, and the Stanford Schizophrenia Genetics Research Fund establishedby an anonymous donor. C.E.A. was supported by California Institute ofRegenerative Medicine Training Grant and the Siebel Foundation. M.W. isa NYSCF–Robertson Stem Cell Investigator, a Howard Hughes Medical Insti-tute Faculty Scholar, and a Tashia and John Morgridge Faculty Scholar. T.C.S.is a Howard Hughes Medical Institute Investigator.

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