Transdifferentiation of Human Adult Peripheral Blood T Cells Into Neurons

Transdifferentiation of human adult peripheral blood T cells into neurons

Koji 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 and Howard 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 conditions, and their growth and formation is labor-intensive (iPS) cells have proven to be powerful new assets for investigating and difficult to scale from a large number of individuals. disease mechanisms. New insights have been obtained studying Another way to obtain neurons is by deriving induced neuro- single mutations using isogenic controls generated by gene nal (iN) cells from fibroblasts in a single conversion step, which targeting. Modeling complex, multigenetic traits using patient- in principle would greatly facilitate their derivation from many derived iPS cells is much more challenging due to line-to-line patients (8). However, unlike neonatal human fibroblasts, adult variability and technical limitations of scaling to dozens or more human fibroblasts have proven difficult to reprogram into syn- patients. Induced neuronal (iN) cells reprogrammed directly from aptically competent iN cells (9–14). Moreover, fibroblasts are dermal fibroblasts or urinary epithelia could be obtained from heterogeneous and ill-defined and must be expanded in vitro many donors, but such donor cells are heterogeneous, show from invasive and painful skin biopsies to obtain sufficient interindividual variability, and must be extensively expanded, numbers, increasing the risk of acquiring random genetic muta- which can introduce random mutations. Moreover, derivation of tions during an extended culture period. Here we report that dermal fibroblasts requires invasive biopsies. Here we show that functional synapse-forming human iN cells can be induced from human adult peripheral blood mononuclear cells, as well as freshly isolated and stored adult peripheral T cells using non- NEUROSCIENCE defined purified T lymphocytes, can be directly converted into integrating episomal vectors. Previous studies have shown the fully functional iN cells, demonstrating that terminally differen- conversion of blood and urinary cells into various neural pro- tiated human cells can be efficiently transdifferentiated into a genitor cells that only inefficiently gave rise to functional neu- distantly related lineage. T cell-derived iN cells, generated by non- – integrating gene delivery, showed stereotypical neuronal morphol- rons (15 21). The described conversions were accomplished with ogies and expressed multiple pan-neuronal markers, fired action transient expression of iPS cell reprogramming factors, an potentials, and were able to form functional synapses. These cells were stable in the absence of exogenous reprogramming factors. Significance Small molecule addition and optimized culture systems have yielded conversion efficiencies of up to 6.2%, resulting in the Recent advances in genomics have revealed that many poly- generation of >50,000 iN cells from 1 mL of peripheral blood in a genetic diseases are caused by complex combinations of many single step without the need for initial expansion. Thus, our common variants with individually small effects. Thus, building method allows the generation of sufficient neurons for experi- informative disease models requires the interrogation of many mental interrogation from a defined, homogeneous, and readily patient-derived genetic backgrounds in a disease-relevant cell accessible donor cell population. type. Current approaches to obtaining human neurons are not easy to scale to many patients. Here we describe a facile, one- induced neuronal cells | direct conversion | transdifferentiation | step conversion of human adult peripheral blood T cells directly disease modeling | iN cells into functional neurons using episomal vectors without the need for previous in vitro expansion. This approach is more dvances in cell reprogramming and genome editing tools amenable than induced pluripotent stem cell-based approaches Ahave provided new ways to interrogate human gene function for application to larger cohorts of individuals and will enable in various human cellular contexts, such as neurons. In particu- the development of functional assays to study complex human lar, genetic engineering of embryonic or induced pluripotent brain diseases. stem (iPS) cells has proven powerful for dissecting the specific consequences of disease-associated mutations in controlled ge- 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; netic backgrounds (1, 2). However, these methods cannot be 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. wrote expected to provide fully adequate cellular models of diseases for the paper. which highly polygenic mechanisms underlie risk. For example, Reviewers: T.G., Center for Genomic Regulation; and H.O., Keio University School large-scale genome-wide association study data suggest that 30– of Medicine. 50% of the genetic risk for each of the neuropsychiatric disorders The authors declare no conflict of interest. that have been studied to date can be explained by the joint Published under the PNAS license. effects of thousands of common genetic variants with small in- Data deposition: The sequences reported in this paper have been deposited in the Gene dividual effects, such that individual patients are likely to be Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. carrying a unique combination of many contributory variants (3). GSE113804). One way to study such complex genetic backgrounds in human 1K.T. and C.E.A. contributed equally to this work. neurons is by reprogramming patient cells to iPS cells (4). 2To whom correspondence may be addressed. Email: [email protected] or wernig@ However, iPS cells have significant line-to-line variability in stanford.edu. terms of differentiation capacity, presumably due to variations in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. their epigenetic and pluripotent state (5–7). Moreover, iPS cells 1073/pnas.1720273115/-/DCSupplemental. are often karyotypically unstable when grown in feeder-free

www.pnas.org/cgi/doi/10.1073/pnas.1720273115 PNAS Latest Articles | 1of6 Downloaded by guest on September 26, 2021 A mouse glia cells and human fibroblasts, but on no other sub- blood cell medium strate. Cells transfected with EGFP alone did not attach to any +IL2 +IL2 N3 medium substrate (SI Appendix, Fig. S1 B and C). On day 5, we changed +Activ. D0 D3 D5 D7 D14 D21 ElectroporationSeeding onmedia Glia changemedia change media change media change (IL2, activator) (BAMN+GFP) A Cont-TUJ1 Cont-MAP2

B C Day1 Day14 5 3sm-TUJ1 3sm-MAP2

2.5

Day21 Day42 0 B 15 C Day 21 Day 42 Relative Number of iN cells

N3 * -IL2 -Activ. Cont +IL2 -Activ. 10 +IL2 +Activ.

* 20mV D EFG2.0 * (n = 3/7) (n = 5/6) 0.8 1.5 Day 21 1.5 Day 21 Day 21 5

Fold-increase 10mS 1.5 * 3sm 1 1 induction efficiency 0 0.4 1 Fo SB Do

3sm (n = 5/9) (n = 5/8) D Cont of iN cells 0.5 0.5 Na+ and K+ channel currents (N3+3sm condition, Day 42)

efficiency (%) 0.5 * electroporation Relative number Transdifferentiation

Relative efficiency of 1.5 + Induction efficiency (%) * Na 0 0 0 0 1 K+ 020406080 o o o o o o 4 C 4 C Age 4 C 0.5 -80 C -80 C Fresh Fresh -80 C Fresh 0 Current (nA) -0.5 Fig. 1. Generation of neuronal cells from peripheral blood cells. (A)Experi- mental outline of iN cell induction from PBMCs. (B) Morphological changes 0.5nA 0.5nA 1ms -80 -40 0 40 during iN cell induction from PBMCs. (Scale bars: 50 μm.) (C) The relative 25ms Voltage (mV) number of iN cells from T cells with or without T cell activator (anti-CD3/CD28), Intrinsic membrane properties Action potential properties with or without IL-2, or a change to N3 media on day 3. n = 3individuals.(D) EF (N3 + 3sm condition) (N3 + 3sm condition) Efficiency of iN cell induction of transduced cells from 35 individual donors * ** * without inhibitors at day 21. n = 1 for each donor. The number of iN cells on -50 * 12 70 -30 + 2 day 21 was divided by the number of total EGFP cells counted on day 1. (E and -40 ns F) Relative iN cell induction (E) and efficiency of electroporation (F)from 8 (mV) 1 (pF) 60 -20 − m

-30 (GOhm) PBMCs of three individual donors that were kept at 80 °C or at 4 °C for 2 d rest 4 C m < V relative to the fresh sample. *P 0.05, paired t test. (G) Transdifferentiation R -20 0 0 50 -10 efficiency of PBMCs from three individual donors kept at −80 °C or at 4 °C for AP height (mV)

2142 2142 21 42 21 42AP threshold (mV) 21 42 2 d relative to the fresh sample. Error bars represent SD. Days Days Days Days Days

Fig. 2. Small molecule treatment improves iN cell conversion efficiency and approach recently shown to induce a pluripotent intermediate maturation. (A) Immunofluorescence analysis of iN cells with and without state (22). small molecules (3sm, three small molecules: forskolin, dorsomorphin, and SB431542; Cont, DMSO). (Scale bars: 50 μm.) (B) Fold change of improved iN Results cell formation on day 21 following various small molecule treatments as indicated. Do, dorsomorphin; Fo, forskolin; SB, SB431542. Data shown are av- Direct Induction of iN Cells from Peripheral Blood Mononuclear Cells. erage fold changes of three independent experiments using PBMCs from To investigate whether blood cells can be transdifferentiated to three different donors. The fold change over the control condition was plotted iN cells, we collected fresh blood from an adult healthy indi- because the absolute reprogramming efficiency was variable among the three do- vidual. We then isolated peripheral blood mononuclear cells nors, but the fold change was consistent. *P < 0.05, paired t test. The error bars (PBMCs) using gradient centrifugation and electroporated these indicate SDs. Similar results were obtained with PBMCs from another set of three cells with episomal vectors encoding the four transcription fac- different donors. (C) Example traces of action potential firing recorded from PBMC- tors Brn2, Ascl1, Myt1l, and Ngn2, collectively termed the derived iN cells with or without 3sm at days 21 and 42 (n represents the number of BAMN pool, which was previously found to generate iN cells cells patched that shows action potentials over the total number of cells patched). from human fibroblasts (9), and enhanced green fluorescent The experiment was performed with cells from three different donors, yielding similar results. (D) Sample traces (Left) and average values (mean ± SEM; Right) protein (EGFP) into 3 million PBMCs. Transfected cells were + + demonstrating the presence of voltage-gated Na and K channels in blood iN cells then cultured in IL-2 and CD3/CD28 antibodies containing cocultured with glia with 3sm for 42 d. (Inset) Expanded view of the dotted boxed media supporting T cell growth (Fig. 1A). On day 3, we placed area. (E) Intrinsic properties of membrane potential (Vrest), capacitance (Cm), and the nonadherent, transfected blood cells on different substrates, input resistance (Rm) approach more mature values over time. (F)Maturationof including primary mouse glia, mouse fibroblast SNL cell line, blood iN cells on extended coculture with glia in 3sm from day 21 to day 42 as human primary fibroblasts, Matrigel, Polyornithine, or laminin- determined by increased action potential (AP) height and threshold. Bar graphs coated dishes. BAMN-transfected cells attached well on primary represent mean ± SEM. *P < 0.05; **P < 0.01. ns, not significant.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1720273115 Tanabe et al. Downloaded by guest on September 26, 2021 the hematopoietic medium to the neuronal medium N3. Re- markably, after seeding of the human blood cells on murine glial cells, we noticed that glial cells deteriorated quickly. We reasoned that the nontransfected, activated human T cells presumably be- A Day 21 B gan to attack the mouse glia. Withdrawal of IL-2 and the T cell TUJ1 (+) 30 10 activators after day 3 not only mitigated this problem, but also MAP2 (+) * * * improved reprogramming by 2.7- to 3.6-fold. Switching to neuro- 20 nal media on day 3 also rescued glial viability but improved 5 reprogramming only slightly (Fig. 1C and SI Appendix,Fig.S1E). 10 * * To test the general applicability of our protocol, we obtained of iN cells blood from a total of 35 healthy adult donors of various ages and Relative number 0 0 ethnicities and both sexes. Surprisingly, the electroporation Relative length neurites SP SP SU SU RA RA efficiencies varied significantly (SI Appendix, Fig. S1A), but Cont Cont IWP2 IWP2 DAPT DAPT Rock i Rock i DMSO DMSO nonetheless we were able to generate morphologically complex 3sm 3sm iN cells from all tested blood samples (Fig. 1D). The reprog- C Cont 3sm 3sm+Rock i ramming efficiency varied as well, but the electroporation rate did not correlate with the reprogramming efficiency (SI Appen- dix, Fig. S1D). Unlike iPS cell reprogramming, reprogramming to iN cells was consistently lower in aged donors (Fig. 1D). Because most biorepositories freeze patient samples, we next D tried to induce iN cells from short- and long-term stored PBMCs 3sm + Rock i E 30 at cold temperature. We isolated PBMCs from a fresh whole- * * blood sample, reprogrammed a fraction of the cells, and froze 20 the remaining cells at −80 °C. Another fraction of the whole- blood sample was maintained at 4 °C for 2 d before subsequent * PBMC isolation. We then reprogrammed the PBMCs isolated EGFP 10 of iN cells from the stored blood sample and the frozen PBMCs. There

Relative Number were no significant differences in the reprogramming efficiency and 0 electroporation efficiency between fresh and frozen PBMCs, but the NEUROSCIENCE 3sm - + + + + + iN cell yield was substantially lower from PBMCs stored at 4 °C due Rock i - - + + + + E–G IWP2 - - - + - + to decreased transfection efficiency (Fig. 1 ). Thus, storage of MAP2 RA - - - - + + freshly isolated cells at −80 °C did not affect reprogramming.

F w/o BDNF and GDNF DMSO Combined BMP and TGF-β Pathway Inhibition and PKA Activation D14 w/ BDNF and GDNF 3sm+Rock i Improved Reprogramming Efficiency. We next sought to further DMSO increase the induction efficiency of iN cells using small mole- D21 3sm+Rock i cules. Blockade of BMP and TGF-β pathways have been shown 3sm+Rock i to promote neural induction during normal development, during – 05 2114 0 612 ES cell differentiation, and from fibroblasts (23 25). Moreover, Relative Number of iN cells Day 21 Day 42 cAMP has been reported to facilitate neuronal survival and G H maturation (26). Therefore, we treated reprogramming blood cells with compounds regulating these three pathways from day DMSO * A 3sm * 5 to day 21 (Fig. 2 ). Indeed, the number of iN cells was sig- (n = 3/7) (n = 5/6) 3sm+Rocki nificantly increased by the adenylyl cyclase activator forskolin Control Day 21 Day 42 3sm+Rocki 3sm (1.9-fold), the BMP pathway blocker dorsomorphin (3.7-fold), and the TGF-β pathway inhibitor SB431542 (4.6-fold) when 0314 210 100 200 300 B 20mV Total no of neurons analyzed on day 21 (Fig. 2 ). The combination of the three in- 10mS hibitors (forskolin, dorsomorphin, and SB431542; 3sm) showed (n = 2/6) (n = 3/6) an additive effect on the number of iN cells relative to the cells 3sm+Rock inhibition seeded (8.7-fold) (Fig. 2 A and B). These combined improve- ments yielded a reprogramming efficiency of up to 6.2%, which Fig. 3. ROCK inhibition improves morphological maturation, but not B functional maturation. (A) Relative number of TUJ1-positive (yellow bars) or translates into 54,265 iN cells from 1 mL of blood (Fig. 2 ). MAP2-positive (blue bars) cells with 3sm (forskolin, dorsomorphin, and SB431542) and additional small molecules from PBMCs from three individual Blood iN Cells Exhibit Passive and Active Neuronal Membrane donors on day 21, normalized to the no treatment control condition (Cont). Properties. We then tested the functional properties of blood n = 3 individuals. *P < 0.05 relative to DMSO (only with 3sm). (B) Relative iN cells that were generated with and without addition of the three average length of neurites with small molecules from PBMCs from three inhibitors. Patch-clamp recordings of blood iN cells showed their individual donors. The length was normalized to the no treatment control ability to fire action potentials on step-current injection at 21 and + (Cont). n = 3 individuals. *P < 0.05 relative to the DMSO. (C) Example pic- 42 d after infection and expressed functional voltage-gated Na + tures of iN cells with indicated inhibitors. Green, EGFP fluorescence (Scale and K channels (Fig. 2 C and D). As expected, 3sm treatment bars: 50 μm.). (D) iN cells generated with 3sm plus ROCK inhibitor express and longer culture periods (day 42) yielded cells with parameters μ neuronal markers, including MAP2. (Scale bars: 50 m.) (E) The effect of more mature action potentials (height, threshold, faster de- of indicated small molecule combinations on reprogramming efficiency. < = polarization and repolarization kinetics) and more mature in- *P 0.05, paired t test. n 3 individuals. (F) The effect of the duration of 3sm trinsic properties, such as increased capacitance and decreased plus ROCK inhibitor with (yellow bars) or without (blue bars) GDNF and = input resistance, compared with control-treated cells and day BDNF on reprogramming efficiency. n 3 individuals. (G) Representative C E F traces of action potential responses of PBMC-derived iN cells under control 21 cells, respectively (Fig. 2 , , and ). condition (N3 only; Top) and in the presence of 3sm plus ROCK inhibitor when cocultured with glia for 21 d (Left)or42d(Right). (H) Graph showing ROCK Inhibition Substantially Improves Formation of Neuronal the total number of neurons in four conditions: DMSO control (black line), 3sm Morphologies but Does Not Improve Functional Properties. In an at- treatment for 21 d (blue line), 3sm plus ROCK inhibitor for 21 d (red line), and tempt to increase the reprogramming efficiencies even further, we 3sm plus ROCK for 14 d and 3sm for 7 d. n = 3individuals.*P < 0.05. screened six additional compounds—IWP2, DAPT, retinoic

Tanabe et al. PNAS Latest Articles | 3of6 Downloaded by guest on September 26, 2021 A BCNeural markers Blood markers PBMC Blood-iN Top significant GO terms PBMC PBMC 500 iN 500 Synaptic transmission iN Chromatin organization MAPK cascade 250 Nervous system development 250 RPKM RPKM Cellular component morphogenesis Intracellular protein transport Upregulated Sensory perception of smell 0 0

CD8 CD3 TAUTUJ1 CD45 D MAP2NCAM E 100 PBMC 100 PBMC DNA recombination iN iN Translation Regulation of cell cycle 50 50

Cell proliferation RPKM RPKM

3.00 Cellular defense response 2.00

1.00 Downregulated Mitosis 0.00 0 0 Fig. 4. Blood iN cells express genes characteristic of -1.00 Defense response to bacterium -2.00 -3.00 P57 excitatory, postmitotic neurons. (A) Heatmap show- MIKI67MCM2CDK1 INK4AINK4BINK4CINK4D ing 6,941 genes differentially expressed between PBMC FGImmunofluorescence H RNA sequencing EGFP VGLUT 150 40 and blood iN cells. Two biological replicates per pop- 35 ulation, greater than twofold change and P < 0.05. 100 30 < 25 Shown are the seven most significant (P 0.05, Bon- 20 ferroni-corrected) Gene Ontology terms among up- and 50 RPKM 15 10 down-regulated genes using PANTHER. (B) Induction of 5 pan-neuronal markers. (C) Suppression of blood cell 0 0

EGFP SATB2 % in Neuronal cells TH genes. (D) Down-regulation of cell cycle activators. (E) SIX3 EN1EN2 TUJ1MAP2 vGAT CTIP2PRPH OTX1 Induction of antiproliferative cyclin-dependent kinase vGLUT GAD65REELIN SATB2 FOXG1 HOXB4HOXC4 IJRNA sequencing RNA sequencing inhibitors. (F) Immunofluorescence of blood iN cells 4 PBMC 40 PBMC showing expression of the excitatory marker vGLUT iN iN and subtype markers SATB2 and CTIP2 at 21 d after infection and cultured with 3sm on glia (Scale bars: EGFP CTIP2 μ 2 20 50 m.). (G) Quantification of day 21 blood iN cells RPKM RPKM grown on glia in 3sm conditions by immunofluorescence for indicated markers. (H) Expression of region- specific markers by the PBMC-derived iN cells by RNA- 0 0 seq. (I) Validation of the neurotransmitter-specific TH markers by RNA-seq. (J) Validation of the cortical sub- DBH RELN CUX1 TBR1SOX5 GAD67 CHAT SATB2 RORBFEZF2CTIP2 VGLUT2 type-specific markers by RNA-seq.

acid, SU5402, Y26732, and SP600125—targeting pathways pre- responses (Fig. 4A). Pan-neuronal markers (TAU, TUBB3, viously implicated in neural differentiation in combination with MAP2, and NCAM) were up-regulated while blood surface forskolin, dorsomorphin, and SB431542. Of the single com- markers (CD8, CD45, and CD3) were silenced (Fig. 4 B and C). pounds tested, we found that ROCK inhibition increased both As expected, proproliferative genes were down-regulated and the relative number of iN cells and the morphological com- negative cell cycle regulators were induced (Fig. 4 D and E). plexity of reprogramming cells compared with the DMSO These results suggest that blood iN cells have silenced hemato- control (Fig. 3 A–D). Other combinations of small mole- poietic transcriptional programs and have adopted a pure neuronal cules did not have additional effects (Fig. 3E). Moreover, the identity. addition of neurotrophic factors did not substantially increase To characterize the regional and neurotransmitter identity of the formation of iN cells, but long-term drug treatment PBMC-derived iN cells, we performed immunofluorescence yielded more iN cells than transient small molecule treatment and considered the results in conjunction with the RNA-seq (Fig. 3F). data. The two methods yielded very consistent results. We Importantly, however, when we tested their functional prop- found that blood iN cells expressed the vesicular glutamate erties, cells generated in the presence of ROCK inhibition were transporter (VGLUT) but not the vesicular GABA transporter, not able to generate mature action potentials, unlike cells grown GAD65, or tyrosine hydroxylase, suggesting that blood iN cells without the ROCK inhibitor (Fig. 3G). The lack of mature action are excitatory neurons similar to fibroblast iN cells and Ngn2- potentials suggests that ROCK inhibition simply affects cyto- ES iN cells (8, 9, 27) (Fig. 4 F, G,andI). Based on the RNA-seq skeletal rearrangements but perturbs functional neuronal matu- results, we found expression of forebrain markers and cortical ration. To assess whether transient ROCK inhibition is sufficient layers II–V but not layers I, IV, and VI (Fig. 4 H and J). Im- to increase conversion efficiency and still allow for functional munofluorescence analysis demonstrated that in fact almost all maturation, we removed the ROCK inhibitor and 2 wk after 3sm blood iN cells were positive for SATB2 and CTIP2 but negative plus ROCK inhibitor treatment and found no beneficial effect for REELIN, as suggested by the RNA results (Fig. 4 F and G). (Fig. 3H). SATB2/CTIP2 double-positive cells are found in layer V of the mouse cortex (28). Molecular Characterization of Blood-Derived iN Cells. To assess the transcriptional changes induced by reprogramming, we per- The Blood-to-Neuron Conversion Does Not Involve a Proliferative formed RNA-sequencing (RNA-seq) on the donor PBMCs as Neural Progenitor State. Several groups have reported the con- + + well as on EGFP and PSA-NCAM blood iN cells purified by version of blood cells into proliferative neural cells using subsets magnetic cell sorting. A total of 6,941 genes were differentially of iPS cell reprogramming factors (20, 21). In contrast, our expressed between the PBMCs and blood iN cells (Fig. 4A). The reprogramming factors are not proproliferative and our ap- up-regulated genes were enriched for Gene Ontology terms such proach yields postmitotic neurons directly. Nevertheless, it may as nervous system development and synaptic transmission, while be possible that even our reprogramming process involves a the down-regulated genes were enriched for cellular defense proliferative intermediate progenitor. To address this question,

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1720273115 Tanabe et al. Downloaded by guest on September 26, 2021 A BCDay 21 transfection protocol (Fig. 1A). We had used a bicistronic de- 1.5 Bright-field CD3+- /CD4 livery of Ngn2 and Ascl1 linked by the T2A self-cleaving peptide. After cleavage, the T2A peptide sequence is predicted to be fused in frame with Ascl1 and thus can serve as a tag of the 1.0 Rec exogenous Ascl1. Immunostaining with T2A antibodies showed that as many as 40% of blood iN cells were T2A-Ascl1 negative, (n = 6/6) and FACS analysis demonstrated that approximately the same

Relative fraction of cells had silenced the EGFP transgene, which was co- ++ Merge CD3 /CD4 electroporated together with the reprogramming factors (SI Appendix, Fig. S3 A and B). Because we used the mouse cDNAs

reprogramming efficiency for all four reprogramming factors to reprogram human cells, we 0 + could accurately distinguish the exogenous (mouse) factors from the endogenous (human) ASCL1, NGN2, BRN2, and MYT1L - -- +- ++ (n = 5/8) 20 mV genes in our RNA-seq dataset. This analysis clearly demon- 20 mS strates that the exogenous factors were effectively silenced in CD3 /CD4 CD3 /CD4 CD3 /CD4 CD3 /CD4 − + the EGFP /PSA-NCAM iN cell population (SI Appendix, DESpontaneous network activity Evoked PSCs Fig. S3C). Thus, blood iN cells can adopt a stable neuronal Control identity without the need for continued expression of exogenous 20 pA reprogramming factors. 2s + Synaptically Competent iN Cells Can Be Derived from CD3 T Cells. PBMCs consist of fairly heterogenous hematopoietic cell pop- 20 pA ulations. We wondered whether iN cells could be established 50 ms from a more defined cell type. After characterizing freshly transfected cells, we found that our electroporation conditions Spontaneous PSCs CNQX+Picrotoxin + Control greatly favor CD3 T cells, suggesting that the vast majority of PBMC iN cells are in fact T cell-derived (SI Appendix, Fig. S4 A and B). To more specifically test whether T cells can be con-

verted, we introduced the BAMN factors into four defined cell NEUROSCIENCE 20 pA populations purified based on CD3 and CD4 expression. Indeed, + − CNQX+Picrotoxin 0.5 s 20pA morphologically complex iN cells were induced from CD3 /CD4 20ms + + − 20 pA and CD3 /CD4 cells, but not from CD3 cells (Fig. 5A). T cell iN cells showed passive and active neuronal membrane properties when cocultured with glia for 18 d and recorded on day 21 (Fig. 5 Fig. 5. Generation of synaptically competent iN cells from T cells. (A) Rel- B C SI Appendix E ative reprogramming efficiency of iN cells from the four indicated PBMC and and , Fig. S4 ). Finally, we confirmed the populations. The plotted efficiency was normalized by the electroporation T cell origin of PSA-NCAM FACS-purified iN cells, by dem- + − β SI efficiency and the efficiency of the CD3 /CD4 cell population was set to 1 onstrating genomic VDJ rearrangements at TCR- locus ( (n = 3 individuals). (B) Recording configuration of EGFP-labeled blood iN cells Appendix, Fig. S4 C and D). cocultured with unlabeled iPS cell-derived neurons. The recording electrode The defining property of a neuron is its ability to make syn- (Rec) was placed onto an EGFP-positive blood iN cell (white arrowhead) aptic connections. Therefore, we asked whether BAMN-induced surrounded by nonfluorescent human iPS cell-derived neurons (black ar- T cell iN cells would be able to form functional synapses. Local rowheads). (Scale bar: 50 μm.) (C) Example traces of action potential firing administration of GABA and AMPA on the soma and proximal recorded from iN cells derived from CD3+/CD4− (red) or CD3+/CD4+ (blue) dendrites of day 21 T cell iN cells recorded in voltage-clamp T cells. N represents the number of cells patched that show action potentials mode yielded prominent GABAA receptor-mediated inhibitory over the total number of cells patched. (D) Representative traces of AMPA postsynaptic currents (PSCs) and AMPA receptor-mediated ex- receptor-mediated spontaneous network activity (Top, black) recorded from citatory PSCs, respectively (SI Appendix, Fig. S4 G and H), a T cell-derived iN cell, indicating successful integration into the human demonstrating the presence of functional neurotransmitter re- synaptic network. The trace in red (Bottom) represents an expanded view of ceptors. To address whether the T cell iN cells have the capacity the boxed area. Spontaneous PSCs recorded from a T cell-derived iN cell to form synapses and functionally integrate into existing neuro- (Top, black) and subsequently blocked by CNQX and picrotoxin (Bottom, nal networks, we plated EGFP-labeled iN cells at 14 d post- black). The trace in red (Middle) represents and expanded view of the boxed area. This pattern was observed in 2 of the 27 cells patched. (E) Evoked PSCs induction onto unlabeled human iPS cell-derived neurons. At (five trials) in response to extracellular field stimulation recorded from a 21 d after coculture, we performed voltage-clamp recordings blood iN cell (Top), which was subsequently blocked by CNQX and picrotoxin from EGFP-positive cells and observed synaptic AMPA receptor- application (Bottom). mediated spontaneous network activities, indicating their successful integration into the synaptic network (Fig. 5D). In addition, spontaneous PSCs could also be observed in these cells, as con- we decided to stain the cells at different time points between firmed by application of the specific AMPA and GABAA receptor days 3 and 21 during reprogramming with the neural progenitor antagonists CNQX and picrotoxin, respectively (Fig. 5D). Finally, marker Sox1 and the proliferation marker Ki67 (SI Appendix, evoked PSCs could also be elicited by activating surrounding axons Fig. S2). To our surprise, Ki67-positive cells decreased only after via extracellular field stimulation of the vicinity (Fig. 5E), unam- the first week of reprogramming. Nonetheless, all Ki67-positive biguously demonstrating that blood iN cells can receive functional cells were Sox1-negative, and the number of proliferative cells synaptic inputs from other neurons. declined rapidly after day 7. Thus, no proper neural progenitor cells are formed as transient intermediates. The initial persis- Discussion tence of proliferative cells is likely due to the cytokines that we Our demonstration that adult human peripheral T cells can be used to activate lymphocytes. directly converted to neurons has both conceptual and practical implications. During normal development, the only cells with the Blood iN Cells Are Stable Without Persistent Transgene Expression. potential to change lineage identity are uncommitted stem and To examine whether the neuronal identity is dependent on progenitor cells. Most reprogramming studies use heterogeneous continued transgene expression, we first examined whether fibroblasts as donor cells, raising the question as to whether the the transgenes were perhaps already silenced in our original transdifferentiation capability is limited to undifferentiated progenitor

Tanabe et al. PNAS Latest Articles | 5of6 Downloaded by guest on September 26, 2021 cells (29). Our results unequivocally show that terminally dif- derived iN cells (20, 21, 30, 31), and using the current protocol, ferentiated cells can be transdifferentiated into another, distantly our blood iN cells are exclusively excitatory. While many cell related somatic lineage. biological processes, such as transcription, polarization, migra- The derivation of neurons from adult peripheral blood cells tion, and subcellular transport, can already be studied in these also has important practical implications. Unlike fibroblasts, cells, future efforts will need to focus on improving synaptic whose derivation requires an invasive and painful skin punch maturation and deriving additional neuronal subtypes. biopsy, lymphocytes can be obtained in large numbers from a simple venipuncture, a procedure performed in almost every Materials and Methods hospitalized patient, often on a daily basis. Moreover, blood PBMCs were isolated from fresh blood donations obtained through the samples are stored in biorepositories in much larger numbers Stanford Blood Bank from individuals of various ethnic backgrounds (Cau- than skin fibroblasts. Of relevance for blood iN cell applications casian, Japanese, Indian, South American, and African), various ages (16– using such repositories, we observed that iN cells can be obtained 78 y), and both sexes using density gradient centrifugation with Ficoll-Paque from fresh and frozen blood cells with similar efficiency. PLUS (GE Healthcare) according to the manufacturer’s instructions. PBMCs Therefore, our blood iN cell conversion described here enables were frozen by a stem cell banker (ZENOAQ). Then 3 μg of vectors (PcxLE- the generation of human neurons from virtually any individual, Ngn2-2A-Ascl1, Brn2, and Myt1L: 0.666 μg; pCXLE-GFP: 0.5 μg; pCXWB- unlike the use of fibroblasts as donor cells, which have proven Ebna1: 0.5 μg) or nonreplicative vectors (pCXWB-Brn2, Ascl1, v5Myt1l, difficult to obtain from certain populations, such as children and flagNgn2 and GFP: 0.5 μg) were electroporated into 3 × 106 isolated PBMCs mentally ill persons. In addition, the greater accessibility allows with the Nucleofector 2b Device (Lonza) with the Amaxa Human T-Cell for scalability of donor individuals, which will be instrumental in Nucleofector Kit, program V-024 (Lonza). – assessing how common, low-risk conferring genetic variants Transduced cells were cultured for 3 d in six-well plates in X-VIVO contribute to cellular function in complex genetic diseases. An- 10 medium (Lonza) supplemented with 30 U/mL IL-2 (PeproTech) and 3.4 μL/mL other advantage over fibroblasts as donor cells is that fibroblasts Dynabeads Human T-Activator CD3/CD28 (Life Technologies). At 3 d after need to be expanded in vitro to obtain sufficient numbers, which electroporation, 0.1–1 × 106 transduced cells were seeded on primary glia may lead to accumulation of deleterious mutations. culture in a well of a 12-well plate. Glia (1.5 × 105) were seeded in a well of a From a mechanistic standpoint, it was unexpected to find that— 12-well plate coated with Matrigel (Corning). At 2 d after seeding, the medium unlike iN cell transdifferentiation from fibroblasts—the early was replaced with DMEM/F12 (Invitrogen) including N2 supplement (Gibco), coculture of glia was critical for transdifferentiation of blood B27 supplement (Gibco), and insulin (5 μg/mL; Sigma Aldrich). The medium cells. The effect of glia seems to be fundamentally different here was changed every 7 d. The RNA-seq files are available in the National than in fibroblast reprogramming, where glial coculture does not Center for Biotechnology Information’s Gene Expression Omnibus database substantially impact conversion efficiency rather than synaptic (accession no. GSE113804). Virus generation, electrophysiology, RNA-sequencing, maturation (8, 9). In contrast, the role of glial factors affected TCR recombination and generation of human embryonic stem cell-derived the generation of iN cells in general when transdifferentiated neurons are described in detail in SI Appendix. from blood cells. Since transfected PBMCs also attached onto a layer of fibroblasts but did not reprogram, we assume that the monolayer ACKNOWLEDGMENTS. We thank Dr. Keisuke Okita, Dr. Kazutoshi Takahashi, of glial cells provides secreted or cell contact-dependent factors and members of the M.W. laboratory for important suggestions. This project to the blood cells that are essential for transdifferentiation in was supported by National Institutes of Health Grants R01 MH092931 and addition to enabling their attachment. U19 MH104172, the New York Stem Cell Foundation (NYSCF)–Robertson While this paper provides a clear proof of concept that human Prize, and the Stanford Schizophrenia Genetics Research Fund established by an anonymous donor. C.E.A. was supported by California Institute of adult peripheral T cells can be converted to iN cells with all key — Regenerative Medicine Training Grant and the Siebel Foundation. M.W. is biochemical and functional properties of neurons, we note that a NYSCF–Robertson Stem Cell Investigator, a Howard Hughes Medical Insti- similar to human fibroblast iN cells—these cells exhibit less mature tute Faculty Scholar, and a Tashia and John Morgridge Faculty Scholar. T.C.S. synaptic properties compared with primary mouse or iPS cell- is a Howard Hughes Medical Institute Investigator.

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