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Exosome-Mediated Ultra-Effective Direct Conversion of Human Fibroblasts into Neural Progenitor-Like Cells Yong Seung Lee, Woon Yong Jung, Hyejung Heo, Min Geun Park, Seung-Hun Oh, Byong-Gon Park, and soonhag kim ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b08297 • Publication Date (Web): 20 Feb 2018 Downloaded from http://pubs.acs.org on February 21, 2018

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ACS Nano is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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TOC 90x42mm (300 x 300 DPI)

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Figure 1 321x356mm (300 x 300 DPI)


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Figure 2 321x395mm (300 x 300 DPI)


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Figure 3 320x580mm (300 x 300 DPI)


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Figure 4 263x455mm (300 x 300 DPI)


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Exosome-Mediated Ultra-Effective Direct Conversion of Human Fibroblasts into Neural Progenitor-Like Cells

Yong Seung Lee†, ‡,*, Woon Yong Jung§*, Hyejung Heo†, ‡, Min Geun Park⊥, Seung-Hun Oh¶, Byong-Gon Park∥, and Soonhag Kim†, ‡,**

†

Institute for Bio-Medical Convergence, College of Medicine, Catholic Kwandong University,

Gangneung-si, Gangwon-do, 270-701, Republic of Korea ‡

Catholic Kwandong University International St. Mary’s Hospital, Incheon Metropolitan City,

404-834, Republic of Korea §

Department of Pathology, Catholic Kwandong University International St. Mary’s Hospital,

Incheon Metropolitan City, 404-834, Republic of Korea ⊥

Department of Surgery, Catholic Kwandong University International St. Mary’s Hospital,

Incheon Metropolitan City, 404-834, Republic of Korea ¶

Department of Neurology, CHA Bundang Medical Center, CHA University, Seongnam,

Republic of Korea ∥

Department of Physiology, College of Medicine, Catholic Kwandong University,

Gangneung-si, Gangwon-do, 270-701, Republic of Korea

*

These authors contributed equally to this work.

**

Corresponding author:

Soonhag Kim, PhD †

Institute for Bio-Medical Convergence, College of Medicine, Catholic Kwandong University,

Gangneung-si, Gangwon-do 270-701, Republic of Korea


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‡

Catholic Kwandong University International St. Mary’s Hospital, Incheon Metropolitan City,

404-834, Republic of Korea E-mail: [email protected]; Telephone: +82-32-290-2771


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ABSTRACT Exosomes, naturally secreted nanoparticles, have been introduced as vehicles for horizontal transfer of genetic material. We induced autologous exosomes containing a cocktail of reprogramming factors (‘reprosomes’) to convert fibroblasts into neural progenitor cells (NPCs). The fibroblasts were treated with ultrasound and subsequently cultured in neural stem cell medium for one day to induce the release of reprosomes composed of reprogramming factors associated with chromatin remodeling and neural lineage-specific factors. After being treated with reprosomes, fibroblasts were converted into NPCs (rNPCs) with great efficiency via activation of chromatin remodeling, so quickly that only five days were required for the formation of 1,500 spheroids showing an NPC-like phenotype. The rNPCs maintained self-renewal and proliferative properties for several weeks and successfully differentiated into neurons, astrocytes, and oligodendrocytes in vitro and in vivo. Reprosome-mediated cellular reprogramming is simple, safe, and efficient to produce autologous stem cells for clinical application.

KEYWORDS: exosome, fibroblast, cellular reprogramming, chromatin remodeling, neural progenitor


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Cell replacement therapy using functional somatic cells is a hot issue in regenerative medicine. One of the major interests in this area is to efficiently and safely obtain customized functional cells from patients. There are two main approaches to generating cells of interest: cellular reprogramming through induced pluripotent stem cell (iPSC) status and direct conversion of adult somatic cells into other functional types. Compared with iPSC differentiation, direct conversion seems to be more promising for clinical use, because it requires relatively simple procedures that bypass most developmental stages, has a short processing time, and has no risk of tumorigenesis associated with pluripotency.1,2 For example, direct conversion of somatic cells into neural progenitor cells (NPCs) has emerged as a useful resource for restoring damaged neural tissue. Induction of NPCs from human dermal fibroblasts (HDFs) has been conducted with the integration of exogenous proneuronal transcription factors with/without chemical compounds or only with a cocktail of chemical compounds.2-6 Although the direct conversion method seems to be favorable for cellular repair, the protocols for using xenobiotics still pose some challenges for clinical application, including insufficient efficiency, largely due to the selective permeability of cellular membranes during the transmembrane transportation of exogenous material and safety concerns about viral vectors or chemical compounds.7,8 To resolve these problems, exosomes, self-made natural transporters capable of safely transferring cocktails of endogenous material, might be an alternative for efficient cellular reprogramming into NPCs. Exosomes are a kind of naturally secreted nano-vesicles measuring between 30 and 200 nm in diameter.9 They are integral nanocarriers containing endogenous genetic information of their originating cells. After the discovery of their role as vehicles for the horizontal transfer of mRNA to elicit phenotypical changes in target cells, several studies have revealed the functions of exosomes in cell differentiation and reprogramming.10 For this type of research,


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stem/progenitor cells, which usually require a meticulous process for separation and enrichment, were used as providers of exosomes or as subjects of exosome-induced differentiation or phenotypic change.11-13 If we can induce the secretion of exosomes containing a cocktail of cellular reprogramming factors for an intended purpose (‘reprosome’) from a patient’s somatic cells, which are easily obtainable, this would be great progress in the clinical application of cellular reprogramming. In this study, we developed an efficient method for inducing NPCs from HDFs through the reprosome-mediated direct conversion of HDFs into NPCs (rNPCs) by treating HDFs with pro-neural reprosomes released from HDFs that were stimulated by ultrasound and cultured in neural media.

RESULTS Exosome release is activated by stress conditions such as heat shock, hypoxia, oxidative stress, and mechanical stress.14-17 We used ultrasound to stimulate exosome release caused by cellular stress. To induce the release of pro-neural exosomes, HDFs were exposed to an ultrasound stimulus (1 W/cm2) for 5 sec and then cultured in human neural stem cell (hNSC) media for one day (UHDF group), while non-treated HDFs (NHDF group) were cultured in hNSC media or fibroblast media (Dulbecco’s modified Eagle’s medium; DMEM). After one day of culture, CD63, a specific marker for exosomes, indicated a conspicuously increased number of multivesicular bodies in the UHDFs compared to the NHDFs (Figure S1a).17 The induced exosomes from the culture media of the UHDFs (iExo) and the null exosomes from the NHDFs (nExo) were isolated using the ultra-filtration method as described previously and showed the typical vesicular morphology of exosomes in transmission electron microscopy images (Figure 1a).10 Western blot analysis confirmed that the expression of CD63 was higher in iExo than in nExo (Figure S1b). On nanoparticle


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tracking analysis, the particle size distribution of iExo ranged about from 50 to 200 nm, with an average of 155.6 ± 4.2 nm (Figure 1b,c). The number of particles ranging from 50 to 200 nm from iExo was 9 × 108, which was 3.2-fold larger than that of nExo (Figure S1c and Movie S1 to S3). These findings suggest that ultrasound can efficiently induce exosome release from HDF. Exosomes, messengers of intercellular communication, are integral vehicles containing the endogenous genetic information of cells.10 To analyze the contents of iExos, the concentrations and qualities of total RNA and microRNA in isolated exosomes were measured by an Agilent 2100 Bioanalyzer, and the numbers of protein coding genes were counted by exosomal RNA sequencing (RNA-Seq). The concentration of total RNA in iExo was 5.3-fold higher than that of nExo (Figure S1d), and the number of read counted genes in iExo was 8400, which was about 4.7-fold greater than that of nExo (1762 genes) (Figure S1e and Table S1). Public database searching and gene ontology analysis revealed that a number of mRNAs in iExo were associated with neurogenesis (Figure 1d and S1g and Tables S2 and S3). Increased expression levels of NPC-specific marker genes in iExo, including Sox1, Sox2, Pax6, and Nestin, were confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR; Figure 1e).3 Among the non-coding small RNAs contained in the exosomes, the proportion of microRNA (miRNA) in iExo (60.57%) was much higher than that in nExo (8.52%) (Figure S1h). In addition, iExo contained 53 out of 72 total neuronal lineage-specific miRNAs (73.6%) reported by Parsons et al., (Figure 1f,g and Table S4).18 The concentration of total protein in iExo was increased by about 20-fold compared to that of nExo (Figure S1i). Immunofluorescence staining and flow cytometry analysis demonstrated that the iExos contained the NPC-specific marker proteins Sox1, Sox2, Pax6, and Nestin (Figure 1h and S1j,k). These findings demonstrated that UHDFs can be induced to release reprosomes containing enriched mRNAs, miRNAs, and proteins associated with


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neurogenesis. A dramatic increase in the numbers of transcripts and proteins in reprosomes induced from UHDFs could be a critical factor for cellular reprogramming, as a large number of genes are expressed in stem cells to sustain differentiation potency.12 To verify that cellular reprogramming for neural phenotypes can be transmitted by reprosomes (Figure 2a), we first investigated whether iExos can be delivered into HDFs. The iExos were first labeled by a lipophilic tracer, DiD (DiD-labeled iExo)11 and further incubated with HDFs for one day. The DiD-labeled iExos were observed in the cytoplasm of the iExo-treated HDFs (Figure 2b, left panel). Additionally, to confirm the transfer of endogenous reprogramming factors by iExos, intact HDFs were first transfected with Cy5.5labeled poly(A)27 (poly(A)27-Cy5.5), and this was followed by the procedure of generating UHDFs to induce the release of engineered exosomes packaging poly(A)27-Cy5.5 (Cy5.5-exo) (Figure S2a). After HDFs were co-cultured with iExos containing Cy5.5-exo for one day, Cy5.5-exos were detected in the cytoplasm of the HDFs (Figure 2b; right panel and Figure S2b and Movie S4). Interestingly, Pax6 expression was induced from these iExo-treated HDFs 24 hrs after treatment. These results demonstrate a great efficiency for the delivery of reprosomes into HDFs. Next, we tested the capability of iExo for cellular reprogramming. HDFs (1 × 105 cells, passage 7) were exposed to exosomes isolated from culture media in which hNSC, NHDF, and UHDF were cultured for one day (hNSC-Exo, nExo, and iExo, respectively). Interestingly, during five days of co-culturing HDFs with the three types of exosomes, only the iExo-treated cells (iExo-HDFs) showed the emergence of compact cell colonies (> 100 µm in diameter) (Figure 2c and S2c,d) and an increase in the expression levels of NPCspecific markers from day 1 (Figure 2d-f and S2e,f). When treated cells with 20 × 1011 iExos/ml, iExo-HDFs showed the highest level of spheroid formation and expression levels of NPC-specific markers (Figure S2g-j). We used this concentration for the subsequent tests


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of the reprogramming potential of iExo. After five days, the number of iExo-HDF colonies reached about 1,500 (Figure S2h), and NPC-like properties, namely increases in the expression levels of NPC-specific genes and proteins, such as Sox1, Sox2, Pax6, and Nestin, were comparable to those seen in hNSCs (Figure 2d,e). Finally, double-positive flow cytometry analysis showed increases in Pax6/Nestin, Sox1/Nestin, and Sox2/Nestin of 74.7%, 68.5%, and 75.8%, respectively, in iExo-HDFs at day 5 (Figure 2f). RNA-Seq analysis showed that the neural specific gene-expression profile of iExo-HDFs was similar to that of hNSCs but distinct from that of HDFs (Figure 2g and Table S5), and gene ontology analysis suggested that many of the over-expressed genes in the iExo-HDFs were associated with neurogenesis (Figure S3a and Table S6). These results suggest that iExo is a reprosome capable of inducing rapid cellular reprogramming, producing high yields of NPC-like cells within five days from HDFs. The iExo-HDF acquired NPC-like properties at day 5 was designated as ‘rNPC’ and the compact colonies of iExo-HDF were defined as ‘rNPC passage 1 (p1).’ Spheroids from rNPC p1 were collected and cultured over several passages to obtain a homogenous population. NPC-specific marker genes and proteins were analyzed, and the rNPCs of p2, p4, p6, and p10 expressed Sox1, Sox2, Pax6, and Nestin at high levels (Figure S3b,c). The Ki-67 immunofluorescence staining demonstrated that these rNPCs are highly proliferative (Figure 2h and S3e).3 The proliferative activity of the rNPCs of p6 was maintained for several weeks (Figure 2i). We also used flow cytometry analysis to demonstrate that the double-positive rates of rNPC p10 for Sox1/Nestin, Sox2/Nestin, and Pax6/Nestin were approximately 88.3%, 78.7%, and 88.9%, respectively (Figure S3e). Together, these results demonstrated that a homogenous expandable population consistently expressed Ki-67 and NPC markers over several passages, which suggests that rNPCs possess the proliferative and self-renewal properties of NPCs.4


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To explain this rapid cellular reprogramming by reprosomes, we focused on the epigenetic regulation of gene expression induced by cellular stress. Interestingly, the iExos contained mRNAs and proteins associated with histone modification and mitogen-activated protein kinase (MAPK) pathway-associated genes (Figure 3a and S4a,b and Table S7). We investigated whether iExos can stimulate the MAPK signaling pathways of target cells, resulting in chromatin remodeling.19 Western blot and flow cytometry analyses demonstrated abruptly increased expression of p38, Erk and Msk1 proteins in HDFs from one day after iExo treatment (Figure 3b,c and S4c). Additionally, the treatment of SB203580 and/or U0126 into iExo-HDFs, which are inhibitors of p38 and Erk in MAPK signaling pathway20,21 led to a significantly decreased expression of each target protein and downstream protein Msk1, as well as NPC-specific genes and proteins, such as Sox1, Sox2, Pax6, and Nestin (Figure S4dg). These data indicate that MAPK signaling pathway is one of key mechanisms for reprosome-mediated fast chromatin remodeling in generating rNPC. Then, we tracked the local chromatin density and histone modification changes in the nuclei of iExo-treated HDFs for three days. We marked the local chromatin density by transfection with H2B protein tagged with green fluorescent protein (H2B-GFP). After iExo treatment, the H2B-GFP localization in the HDF nuclei became loose with time (Figure 3d and Movie S5).22 Likewise, the nuclear expression levels of heterochromatin protein 1 α (HP1α) and the repressive histone modification H3K27me323 were decreased, and that of the activating histone modification H3K4me324 were increased with time after iExo treatment of HDFs (Figure 3e-g and S4h,i). Notably, the DNA methylation profiling for neurogenesis-associated genes of rNPCs exhibited lowered methylation (Figure 3h and Table S8). These results indicate that reprosomes can mediate rapid cellular reprogramming into NPC-like cells by activation of the MAPK kinase signaling pathway and chromatin remodeling as well as several epigenetic regulators. Finally, chromosomal G-band analysis of rNPCs showed a normal karyotype of


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46XY (Figure S4j). To evaluate the neural differentiation potency of rNPCs, we examined whether they can differentiate into neural lineages such as neurons, astrocytes, and oligodendrocytes. We collected rNPC spheroids on day 5 and then cultured them on gelatin-coated tissue plates according to a recently reported protocol25,26 for neural differentiation. Four weeks after neural differentiation, the expression of Map2 and Tuj1 (neuronal markers), Gfap (astrocyte marker), and O4 (oligodendrocyte marker) was observed in the differentiated cells (Figure 4a).3 The qRT-PCR analysis of Map2, Tuj1, Gfap, S100b (astrocyte marker), Mbp (oligodendrocyte marker), and Oligo1 (oligodendrocyte

marker) provided further

confirmation that the rNPCs had successfully differentiated into neural cells (Figure S5a).5 We then investigated functional properties of rNPC-derived neurons by whole-cell patch clamp recordings. First, gene expression related to K+ channel (EAG1, Kv4.3, and Kv7.2) and Na+ channel (Nav 1.3, Nav1.6, and Nav1.7) was highly activated after neuronal differentiation from rNPCs (Figure S5b).27 In current-clamp model, rNPC-derived neurons showed both inward Na+ and outward K+ currents (Figure 4b and S5c-f). Besides, a sodium channel blocker, tetrodotoxin (TTX), inhibited the Na+ current of rNPC-derived neurons (Figure S5g).28 The rNPC-derived neurons triggered spontaneous action potential in response to current clamp (Figure 4c). These results indicate that rNPCs have multipotency, effectively differentiating into neurons, astrocytes, and oligodendrocytes in vitro. To evaluate the multipotent differentiation of rNPCs in vivo, we used HDFs transfected with a cytomegalovirus (CMV) promoter-driven GFP reporter gene and GFP reporter stable cells established by G418 antibiotics (GFP-HDF) (Figure S6a). The GFP-HDF-derived rNPCs (GFP-rNPCs) showed NPC-like features, including neurosphere formation and expression of Sox1, Sox2, Pax6, and Nestin (Figure S6b,c), and the differentiation potency was the same as that of the rNPCs (Figure S6d). The GFP-rNPC spheroids (about 1,500


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clusters) on day 5 were then implanted into the brains (striata) of normal Sprague Dawley rats (n = 5), and we assessed their differentiation status after four weeks. We observed numerous GFP-positive cells in multiple brain sections, many of which had formed long neurites (Figure 4d), indicating effective survival, migration, and host integration. Furthermore, the GFP-positive cells stained well with a human mitochondrial antibody (HuMito) (Figure 4e).7 Some of the Gfap+ astrocytes, Map2+ and Tuj1+ neurons, and O4+ oligodendrocytes showed GFP-positivity (Figure 4f). These results clearly demonstrated that engrafted rNPCs, which were induced from HDFs in only five days, have multipotency capable of differentiation into three discrete cell types of neural lineage in vivo.

DISCUSSION Extracellular vesicles (EVs), including exosomes, contain cellular reprogramming factors and can mediate phenotypic change. For example, microvesicles derived from radiation-injured lung cells have induced bone marrow cells to express pulmonary epithelial cell-specific genes and proteins, while exosomes from human umbilical cord mesenchymal stem cells have activated protective programs in injured renal tubular cells, such as inhibition of apoptosis, improvement of cellular proliferation, and reduction of oxidative stress.12,29 However, in these earlier reports, EVs were merely used for directed differentiation of stem cells into functional somatic cells or activation of regenerative programs in injured somatic cells. In this study, ultrasound stimulation of HDFs followed by culturing in NSC media quickly induced reprosome release; furthermore, the induced reprosomes converted HDFs into NPCs very efficiently, so quickly that, in only five days, 1,500 spheroids showing NPClike phenotypes were formed. This was possible due to a cocktail of dramatically increased numbers of reprogramming factors in reprosomes, including mRNAs for neural reprogramming factors such as SOX2, KLF4, MYC, and TCF3 genes,30 epigenetic regulators


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including chromatin remodeling factors such as MAPK/ERK pathway proteins,19 and modified histones and neurogenesis-associated miRNAs such as miR-9, miR-124a, miR-125b, and miR-128-1.31-34 Recently, we reported a method of generating extramultipotent cells which are very similar with pluripotent cells, using ultrasound-directed permeation of environmental transition-guided cellular reprogramming (Entr).35 The Entr cells show extracellular environment-restricted genotype and phenotype in a day. Therefore, a numbers of transcripts and proteins related to neurogenesis and epigenetic regulators in reprosomes might be significantly correlated with the procedure of ultrasound-treated HDFs culturing in hNSC media. In addition with these various reprogramming factors, the phospholipid-based envelopes of the reprosomes were another factor of improved reprogramming efficiency by making it possible for various reprogramming factors to readily cross the plasma membranes of recipient cells, which is the most critical limiting step for gene delivery by non-viral synthetic vectors.36,37 Exosome-mediated transdifferentiation into NPCs is very meaningful since this process involves no gene integration using viral vectors, exogenous transcription factors, or chemical compounds, which are inevitable for cellular reprogramming by existing strategies. In summary, autologous reprosome-induced cellular reprogramming can be a simple and cost-effective solution for practical problems in direct conversion, such as the long duration of cell generation, limited numbers of converted cells, and safety concerns about xenograft or xenobiotics, which are major obstacles for clinical application of existing strategies.


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METHODS

Cell culture. Human dermal fibroblasts and human neural stem cells were purchased from Gibco, Inc. (Gibco, Grand Island, NY, USA); these cells were derived from adult skin and fetal brain, respectively. HDF cells were maintained in DMEM (Gibco) with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco). The hNSCs were suspension cultured in hNSC media of StemPro® NSC SFM (Gibco) supplemented with 2 mM GlutaMAX™-I Supplement (Gibco), 6 U/mL heparin (Sigma-Aldrich, St. Louis, MO, USA), and 200 µM ascorbic acid (Sigma-Aldrich). All cells were cultured at 37 °C in an atmosphere of 5% CO2.

Reprosome isolation and rNPC generation from HDFs. Ultrasound stimulus (UltraRepro 1001, STEMON Inc., Seoul, Republic of Korea) directly exposed to 1 × 106 HDFs (20 KHz, 1.0 W/cm2, for 5 sec, UHDFs). Then, 2 × 105 UHDFs were cultured on a 35-mm petri dish with ultrasound-treated hNSC media (20 KHz, 5.0 W/cm2, for 10 min) for one day. Exosomes were isolated from the culture media of the UHDFs as previously described method.

38

The

culture media was centrifuged at 3,000 × g for 20 min to remove cellular debris and dead cells, and the resulting supernatant was passed through a 0.22-mm filter (Minisart® Syringe Filter, Sartorius, Goettingen, Germany). The filtered medium was transferred to an Amicon® Ultra-15 100,000 kDa device (Millipore, Billerica, MA, USA), and the exosome were concentrated by centrifugation at 14,000 × g for 20 min. To generate rNPCs, 1 × 105 HDFs were seeded and cultured on a 35-mm petri dish for one day. Then, the media was replaced with hNSC media including isolated exosomes and cultured for five days. The culture media was replaced every two days.


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ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at DOI: The Supporting information includes Additional Methods, Supplementary Text for abbreviated words, Figure S1-S6, Tables S9-11, Captions for Tables S1-8, Captions for Movies S1-5, and supplementary references (PDF). Table S1: Results of RNA-Seq analysis for total mRNA expressed in nExo and iExo. (Excel) Table S2: Results of RNA-Seq analysis for neuron-associated gene expression in nExo and iExo. (Excel) Table S3: Gene ontology (GO) analysis of the 2870 genes upregulated more than five-fold in iExo compared to nExo. (Excel) Table S4: Results of RNA-Seq analysis for neuron-related miRNAs in nExo and iExo. (Excel) Table S5: Results of RNA-Seq analysis for neuron-associated gene expression in HDFs, hNSCs, and rNPCs (passages 1 and 5). (Excel) Table S6: Gene ontology (GO) analysis of the 2518 genes upregulated more than 10fold in five-day-old rNPCs compared to HDFs. (Excel) Table S7: Results of RNA-Seq analysis for expression of genes related to histone modification and MAPK signaling pathways in nExo and iExo. (Excel) Table S8: Relative methylation scores in the promoter regions of neural-associated genes from HDFs, hNSCs, and rNPCs. (Excel) Movie S1: Live tracking image of nanoparticle of nExos released from NHDFs cultured in hNSC media as shown by a NanoSight LM10 microscope using a 405-nm laser. (AVI) Movie S2: Dynamic nanoparticle tracking images of nExos released from NHDFs as shown by a NanoSight LM10 microscope using a 405-nm laser. (AVI) Movie S3: Live images of nanoparticle tracking of iExos visualized by a NanoSight LM10 microscope using a 405-nm laser. (AVI) Movie S4: The changes in H2B-GFP expression in the nuclei of HDFs after iExo treatment. (AVI) Movie S5: Delivery of iExos into HDFs. iExos labeled with DiD dye were co-cultured with HDFs. (AVI)


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AUTHOR INFORMATION Corresponding Authors ** E-mail: [email protected] Author Contributions * Y.S.L and W.Y.J contributed equally. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (NRF-2017R1A2B2002042).


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Figure 1. Characteristics of induced exosomes from UHDF. (a) Electron microscope images of exosome morphology from nExos and iExos. Scale bars, 100 nm. (b) Dynamic tracking images of nExo and iExo captured by the NanoSight from Movie S1 to S3. Exosome secretion by ultrasound stimulation was efficient (iExo), contrasted with insignificant exosome secretion from non-stimulated HDFs (nExo) cultured in hNSC media or DMEM for 24 hrs. (c) Particle size distribution analysis of iExos and nExos using NanoSight. (d-g) Expression of neural-associated mRNAs and miRNAs from iExos and nExos. Heatmaps of mRNAs (d) and miRNAs (f) using RNA-Seq analysis. qRT-PCR analysis of mRNAs (e) and miRNAs (g). All quantitative data are represented as the mean ± standard error of triplicated samples. (h) Immunostaining for expression of NPC-specific markers in exosomes from NHDFs and UHDFs at 24 hrs after ultrasound stimulus. White boxes (zoomed exosomes) indicate that NPC marker proteins were packaged in secreted iExos from UHDF. Scale bars, 50 µm.

Figure 2. Generation of neural progenitor-like cells by reprosomes. (a) Scheme of the reprosome-induced direct conversion of HDFs into NPCs. (b) Confocal microscope images for the internalization of iExos into recipient HDFs. iExos labeled by DiD (left panel) or packaging poly(A)27-Cy5.5 (right panel) were incubated with HDFs for 24 hrs. Pax6 expression appeared in recipient HDFs. Scale bars, 50 µm. (c-f) The process of phenotypic changes of HDFs after iExo treatment. Cellular morphologic change from individually scattered spindle cells to conglomerate spheroid of round cells within 5 days after iExo treatment (c, inset: magnified image), gradual increase in NPC specific mRNA expression with time from day-1 (1D) to day-5 (5D) (qRT-PCR) (d), conspicuous expression of NPC specific proteins from day-3 after iExo treatment (immunofluorescent stain) (e, scale bars, 100 µm), gradual increase in cell population expressing NPC specific proteins (flow


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cytometry) (f). (g) Hierarchical cluster analysis of NSC-related genes from HDFs, hNSCs, and rNPCs (at passages 1 and 5). The color bar indicates gene expression. (h) Immunostaining of rNPC p1 for Ki-67. The rNPC p1 is mono cultured cells from compact colonies that acquired NPC-like properties at day 5 by iExo. The cells were cultured at 0.1% gelatin-coated cover slip for immunofluorescence staining analysis. (i) Growth curve of rNPCs. The initial cells were used at passage 6.

Figure 3. Mechanisms involved in generation of rNPCs by reprosomes. (a) Heatmap of iExo and nExo for genes related to histone modification and the MAPK pathway. The color bar indicates gene read counts. Western blot analysis (b) and confocal images (c, scale bars, 50 µm) of phosphorylated Erk1/2, p38, and Msk1 proteins in rNPC for 5 days. For immunofluorescence staining, rNPC spheroids in suspension were collected on the 5th day and cultured in monolayer on the 0.1% gelatin coated cover slip for 12 hrs. (d) Time-lapse images of H2B-GFP expression in iExo-treated HDFs for 72 hrs. A cell indicated by a white arrow is magnified as an inlet. The graph shows the changes in H2B-GFP fluorescence intensity from the cell indicated by the white arrow. (e-g) Immunofluorescence images for chromatin change of rNPC with (e) HP1α, (f) H3K4me3, and (g) H3K27me3 antibodies. Scale bars, 100 µm. White dotted arrows over magnified nuclei (in white box) indicate a line scan of intensity plots. (h) Methylation heatmap of neural-associated genes from HDFs, hNSCs, and rNPCs by MBD-Seq analysis. The color bar indicates the relative methylation score (RMS).

Figure 4. In vitro and in vivo multi-lineage differentiation of rNPCs. (a) Immunostaining of neural markers from in vitro differentiated rNPCs. Scale bars, 100 µm. The rNPCs were cultured in differentiation media for four weeks. (b, c) Whole-cell current-clamp recordings


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of rNPC-derived neurons by patch clamp analysis. (b) Voltage-clamp recording of inward Na+ and outward K+ current on differentiated rNPCs. The red dotted box indicates extended Na+ currents. (c) Representative images of action potential like events recorded on differentiated rNPCs. (d-f) In vivo differentiation of GFP-rNPCs. Distribution (d), immunohistochemical analysis with a human mitochondrial antibody (e), and neural marker expression (f) in GFP-rNPCs at four weeks after implantation into a rat brain. Scale bars, 50 or 100 µm. The zoomed regions from white boxes are shown alongside.


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REFERENCES (1) Li, X.; Zuo, X.; Jing, J.; Ma, Y.; Wang, J.; Liu, D.; Zhu, J.; Du, X.; Xiong, L.; Du, Y.; et al., SmallMolecule-Driven Direct Reprogramming of Mouse Fibroblasts into Functional Neurons. Cell Stem

Cell 2015, 17 , 195-203. (2) Mertens, J.; Marchetto, M. C.; Bardy, C.; Gage, F. H., Evaluating Cell Reprogramming, Differentiation and Conversion Technologies in Neuroscience. Nat Rev Neurosci 2016, 17, 424-437. (3) Yu, K. R.; Shin, J. H.; Kim, J. J.; Koog, M. G.; Lee, J. Y.; Choi, S. W.; Kim, H. S.; Seo, Y.; Lee, S.; Shin, T. H.; et al., Rapid and Efficient Direct Conversion of Human Adult Somatic Cells into Neural Stem Cells by HMGA2/let-7b. Cell Rep 2015, 10, 441-452. (4) Lu, J.; Liu, H.; Huang, C. T.; Chen, H.; Du, Z.; Liu, Y.; Sherafat, M. A.; Zhang, S. C., Generation of Integration-Free and Region-Specific Neural Progenitors from Primate Fibroblasts. Cell Rep 2013,

3, 1580-1591. (5) Ring, K. L.; Tong, L. M.; Balestra, M. E.; Javier, R.; Andrews-Zwilling, Y.; Li, G.; Walker, D.; Zhang, W. R.; Kreitzer, A. C.; Huang, Y., Direct Reprogramming of Mouse and Human Fibroblasts into Multipotent Neural Stem Cells with a Single Factor. Cell Stem Cell 2012, 11, 100-109. (6) Mirakhori, F.; Zeynali, B.; Rassouli, H.; Shahbazi, E.; Hashemizadeh, S.; Kiani, S.; Salekdeh, G. H.; Baharvand, H., Induction of Neural Progenitor-Like Cells from Human Fibroblasts via a Genetic Material-Free Approach. PLoS One 2015, 10, e0135479. (7) Joo, J. Y.; Lee, J.; Ko, H. Y.; Lee, Y. S.; Lim, D. H.; Kim, E. Y.; Cho, S.; Hong, K. S.; Ko, J. J.; Lee, S.; et

al., Microinjection Free Delivery of miRNA Inhibitor into Zygotes. Sci Rep 2014, 4, 5417. (8) Masuda, S.; Wu, J.; Hishida, T.; Pandian, G. N.; Sugiyama, H.; Izpisua Belmonte, J. C., Chemically Induced Pluripotent Stem Cells (CiPSCs): a Transgene-Free Approach. J Mol Cell Biol 2013, 5, 354355. (9) Hannafon, B. N.; Ding, W. Q., Intercellular Communication by Exosome-Derived MicroRNAs in Cancer. Int J Mol Sci 2013, 14, 14240-14269. (10) Quesenberry, P. J.; Aliotta, J.; Deregibus, M. C.; Camussi, G., Role of Extracellular RNA-Carrying Vesicles in Cell Differentiation and Reprogramming. Stem Cell Res Ther 2015, 6, 153. (11) Ratajczak, J.; Miekus, K.; Kucia, M.; Zhang, J.; Reca, R.; Dvorak, P.; Ratajczak, M. Z., Embryonic Stem Cell-Derived Microvesicles Reprogram Hematopoietic Progenitors: Evidence for Horizontal Transfer of mRNA and Protein Delivery. Leukemia 2006, 20, 847-856. (12) Aliotta, J. M.; Sanchez-Guijo, F. M.; Dooner, G. J.; Johnson, K. W.; Dooner, M. S.; Greer, K. A.; Greer, D.; Pimentel, J.; Kolankiewicz, L. M.; Puente, N.; et al., Alteration of Marrow Cell Gene Expression, Protein Production, and Engraftment into Lung by Lung-Derived Microvesicles: a Novel Mechanism for Phenotype Modulation. Stem Cells 2007, 25, 2245-2256. (13) Deregibus, M. C.; Cantaluppi, V.; Calogero, R.; Lo Iacono, M.; Tetta, C.; Biancone, L.; Bruno, S.; Bussolati, B.; Camussi, G., Endothelial Progenitor Cell Derived Microvesicles Activate an Angiogenic Program in Endothelial Cells by a Horizontal Transfer of mRNA. Blood 2007, 110, 2440-2448. (14) Eldh, M.; Ekstrom, K.; Valadi, H.; Sjostrand, M.; Olsson, B.; Jernas, M.; Lotvall, J., Exosomes Communicate Protective Messages during Oxidative Stress; Possible Role of Exosomal Shuttle RNA.


Page 24 of 26

Page 25 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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PLoS One 2010, 5, e15353. (15) King, H. W.; Michael, M. Z.; Gleadle, J. M., Hypoxic Enhancement of Exosome Release by Breast Cancer Cells. BMC cancer 2012, 12, 421. (16) Takeuchi, T.; Suzuki, M.; Fujikake, N.; Popiel, H. A.; Kikuchi, H.; Futaki, S.; Wada, K.; Nagai, Y., Intercellular Chaperone Transmission via Exosomes Contributes to Maintenance of Protein Homeostasis at the Organismal Level. Proc Natl Acad Sci U S A 2015, 112, E2497-E2506. (17) Park, J. A.; Sharif, A. S.; Tschumperlin, D. J.; Lau, L.; Limbrey, R.; Howarth, P.; Drazen, J. M., Tissue Factor-Bearing Exosome Secretion from Human Mechanically Stimulated Bronchial Epithelial Cells

in Vitro and in Vivo. J Allergy Clin Immunol 2012, 130, 1375-1383. (18) Parsons, X. H.; Parsons, J. F.; Moore, D. A., Genome-Scale Mapping of MicroRNA Signatures in Human Embryonic Stem Cell Neurogenesis. Mol Med Ther 2012, 1, 105. (19) Brami-Cherrier, K.; Roze, E.; Girault, J. A.; Betuing, S.; Caboche, J., Role of the ERK/MSK1 Signalling Pathway in Chromatin Remodelling and Brain Responses to Drugs of Abuse. J

Neurochem 2009, 108, 1323-1335. (20) Sukezane, T.; Oneyama, C.; Kakumoto, K.; Shibutani, K.; Hanafusa, H.; Akagi, T., Human Diploid Fibroblasts are Resistant to MEK/ERK-Mediated Disruption of the Actin Cytoskeleton and Invasiveness Stimulated by Ras. Oncogene 2005, 24, 5648-5655. (21) Cuenda, A.; Rousseau, S., p38 MAP-Kinases Pathway Regulation, Function and Role in Human Diseases. Biochim Biophys Acta Mol Cell Res 2007, 1773, 1358-1375. (22) Hinde, E.; Cardarelli, F.; Chen, A.; Khine, M.; Gratton, E., Tracking the Mechanical Dynamics of Human Embryonic Stem Cell Chromatin. Epigenet chromatin 2012, 5, 20. (23) Musselman, C. A.; Lalonde, M. E.; Côté, J.; Kutateladze, T. G., Perceiving the Epigenetic Landscape through Histone Readers. Nat Struct Mol Biol 2012, 19, 1218-1227. (24) Reik, W., Stability and Flexibility of Epigenetic Gene Regulation in Mammalian Development.

Nature 2007, 447, 425-432. (25) Kang, P. J.; Moon, J. H.; Yoon, B. S.; Hyeon, S.; Jun, E. K.; Park, G.; Yun, W.; Park, J.; Park, M.; Kim, A.; et al., Reprogramming of Mouse Somatic Cells into Pluripotent Stem-Like Cells Using a Combination of Small Molecules. Biomaterials 2014, 35, 7336-7345. (26) Yoshikawa, K.; Naitoh, M.; Kubota, H.; Ishiko, T.; Aya, R.; Yamawaki, S.; Suzuki, S., Multipotent Stem Cells are Effectively Collected from Adult Human Cheek Skin. Biochem biophys Res Commun 2013, 431, 104-110. (27) Vacher, H.; Mohapatra, D. P.; Trimmer, J. S., Localization and Targeting of Voltage-Dependent Ion Channels in Mammalian Central Neurons. Physiol Rev 2008, 88, 1407-1447. (28) Victor, M. B.; Richner, M.; Hermanstyne, T. O.; Ransdell, J. L.; Sobieski, C.; Deng, P. Y.; Klyachko, V. A.; Nerbonne, J. M.; Yoo, A. S., Generation of Human Striatal Neurons by MicroRNA-Dependent Direct Conversion of Fibroblasts. Neuron 2014, 84, 311-23. (29) Zhou, Y.; Xu, H.; Xu, W.; Wang, B.; Wu, H.; Tao, Y.; Zhang, B.; Wang, M.; Mao, F.; Yan, Y.; et al., Exosomes Released by Human Umbilical Cord Mesenchymal Stem Cells Protect against CisplatinInduced Renal Oxidative Stress and Apoptosis in Vivo and in Vitro. Stem Cell Res Ther 2013, 4, 34.


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(30) Han, D. W.; Tapia, N.; Hermann, A.; Hemmer, K.; Hoing, S.; Arauzo-Bravo, M. J.; Zaehres, H.; Wu, G.; Frank, S.; Moritz, S.; et al., Direct Reprogramming of Fibroblasts into Neural Stem Cells by Defined Factors. Cell Stem Cell 2012, 10, 465-472. (31) Boissart, C.; Nissan, X.; Giraud-Triboult, K.; Peschanski, M.; Benchoua, A., miR-125 Potentiates Early Neural Specification of Human Embryonic Stem Cells. Development 2012, 139, 1247-1257. (32) Franzoni, E.; Booker, S. A.; Parthasarathy, S.; Rehfeld, F.; Grosser, S.; Srivatsa, S.; Fuchs, H. R.; Tarabykin, V.; Vida, I.; Wulczyn, F. G., miR-128 Regulates Neuronal Migration, Outgrowth and Intrinsic Excitability via the Intellectual Disability Gene Phf6. eLife 2015, 4, e04263. (33) Victor, M. B.; Richner, M.; Hermanstyne, T. O.; Ransdell, J. L.; Sobieski, C.; Deng, P. Y.; Klyachko, V. A.; Nerbonne, J. M.; Yoo, A. S., Generation of Human Striatal Neurons by MicroRNA-Dependent Direct Conversion of Fibroblasts. Neuron 2014, 84, 311-323. (34) Yoo, A. S.; Sun, A. X.; Li, L.; Shcheglovitov, A.; Portmann, T.; Li, Y.; Lee-Messer, C.; Dolmetsch, R. E.; Tsien, R. W.; Crabtree, G. R., MicroRNA-Mediated Conversion of Human Fibroblasts to Neurons.

Nature 2011, 476, 228-231. (35) Lee, Y. S.; Heo, H.; Lee, J.; Moon, S. U.; Jung, W. Y.; Park, Y. K.; Park, M. G.; Oh, S.-H.; Kim, S., An Ultra-Effective Method of Generating Extramultipotent Cells from Human Fibroblasts by Ultrasound. Biomaterials 2017, 143, 65-78. (36) Al-Dosari, M. S.; Gao, X., Nonviral Gene Delivery: Principle, Limitations, and Recent Progress.

AAPS J 2009, 11, 671-681. (37) Seow, Y.; Wood, M. J., Biological Gene Delivery Vehicles: beyond Viral Vectors. Mol Ther 2009,

17, 767-777. (38) Lobb, R. J.; Becker, M.; Wen, S. W.; Wong, C. S.; Wiegmans, A. P.; Leimgruber, A.; Moller, A., Optimized Exosome Isolation Protocol for Cell Culture Supernatant and Human Plasma. J Extracell

Vesicles 2015, 4, 27031.


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