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Transdifferentiation of Human Adult Peripheral Blood T Cells Into Neurons

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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 in- dicated.
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