journal of proteomics · journal of proteomics 165 (2017) 93–101 ⁎...

Journal of Proteomics 165 (2017) 93–101

Contents lists available at ScienceDirect

Journal of Proteomics

j ourna l homepage: www.e lsev ie r .com/ locate / jp rot

Proteomic analysis of mesenchymal to Schwann cell transdifferentiation

Anup D. Sharma a,d, Jayme Wiederin b, Metin Uz a, Pawel Ciborowski b, Surya K. Mallapragada a,d,Howard E. Gendelman b, Donald S. Sakaguchi c,d,⁎a Department of Chemical and Biological Engineering, Iowa State University, Ames, IA 50011-2230, USAb Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198-5880, USAc Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011-1031, USAd Neuroscience Program, Iowa State University, Ames, IA 50011, USA

⁎ Corresponding author at: Department of Genetics, DevScience II, Iowa State University, Ames, IA 50011-1031, U

E-mail address: [email protected] (D.S. Sakaguchi

http://dx.doi.org/10.1016/j.jprot.2017.06.0111874-3919/© 2017 Elsevier B.V. All rights reserved.

a b s t r a c t
a r t i c l e i n f o
Article history:Received 18 April 2017Received in revised form 31 May 2017Accepted 13 June 2017Available online 17 June 2017

While transplantation of Schwann cells facilitates axon regeneration, remyelination and repair after peripheralnerve injury clinical use is limited by cell bioavailability. We posit that such limitation in cell access can be over-come by the use of autologous bone-marrow derived mesenchymal stem cells (MSCs). As MSCs cantransdifferentiate to Schwann cell-phenotypes and accelerate nerve regeneration we undertook proteomic eval-uation of the cells to uncover the protein contents that affects Schwann cell formulation. TransdifferentiatedMSCs secrete significant amounts of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF)in cell-conditioned media that facilitated neurite outgrowth. MSC proteins significantly regulated duringSchwann cell transdifferentiation included, but were not limited to, GNAI2, MYL9, ACTN4, ACTN1, ACTB, CAV-1,HSPB1, PHB2, TBB4B, CTGF, TGFI1, ARF6, EZR, GELS, VIM,WNT5A, RTN4, EFNB1. These support axonal guidance,myelination, neural development and neural growth and differentiation. The results unravel the molecularevents that underlie cell transdifferentiation that ultimately serve to facilitate nerve regeneration and repair insupport of cell transplantation.Significance statement:While Schwann cells facilitate axon regeneration, remyelination and repair after peripher-al nerve injury clinical use is limited by cell bioavailability.Weposit that such limitation in cell access can beover-come by the use of bone-marrow derived mesenchymal stem cells (MSCs) transdifferentiated to Schwann cell-phenotypes. In the present study, we undertook the first proteomic evaluation of these transdifferentiatedcells to uncover the protein contents that affects Schwann cell formulation. Furthermore, thesetransdifferentiated MSCs secrete significant amounts of brain-derived neurotrophic factor (BDNF) and nervegrowth factor (NGF) in cell-conditioned media that facilitated neurite outgrowth. Our results demonstrate thata number of MSC proteins were significantly regulated following transdifferentiation of the MSCs supportingroles in axonal guidance, myelination, neural development and differentiation. The conclusions of the presentwork unravel the molecular events that underlie cell transdifferentiation that ultimately serve to facilitatenerve regeneration and repair in support of cell transplantation. Our study was the first proteomic comparisondemonstrating the transdifferentiation of MSCs and these reported results can affect a wide field of stem cell biol-ogy, tissue engineering, and proteomics.

© 2017 Elsevier B.V. All rights reserved.

Keywords:Schwann cellsAxon regenerationNerve growth factorBrain-derived neurotrophic factorNeurite outgrowthRemyelinationMesenchymal stem cellsTransdifferentiationProteomicsSystems biologyPeripheral nerve regeneration

1. Introduction

Peripheral nerve injuries result in disruption of neural signaling thatoccurs between affected limbs and the central nervous system (CNS)often leading to severe neurological morbidities and even paralysis [1].Anterograde (Wallerian) degeneration initiates a cascade of mixed de-generative and regenerative events initiated by infiltration of peripheralblood derived monocyte-macrophages and end organ Schwann cells

elopment and Cell Biology, 505SA.).

(SCs). The former serve to remove the debris created by degradationof axonal tissue, axonal fibers and the myelin sheath [2] and the latterprovide cells for regenerative activities. SCs present in the distal end ofthe injured tissue effect the secretion of neurotrophic factors. Such fac-tors attract axons regenerating from the proximal endof the injury [3]. Ifsufficient support is provided to the regenerating axonal growth cones,theymay navigate across the nerve gap through a nerve bridge and thengrow into the remaining skeleton of degenerated neural tissue. In moreextreme cases where a large gapmay exist, a range of strategies, are op-erative that provide support for regenerating neural tissue. This is re-ferred to as a “nerve guidance conduit” which can be used tophysically bridge the transected ends of the nerve and as such provide

http://crossmark.crossref.org/dialog/?doi=10.1016/j.jprot.2017.06.011&domain=pdfhttp://dx.doi.org/10.1016/j.jprot.2017.06.011mailto:[email protected]://dx.doi.org/10.1016/j.jprot.2017.06.011http://www.sciencedirect.com/science/journal/18743919www.elsevier.com/locate/jprot

94 A.D. Sharma et al. / Journal of Proteomics 165 (2017) 93–101

support for regenerating axons [4–7]. Nerve guidance conduits filledwith SCs is a promising strategy to promote neural and tissue regener-ation following injury [8–10]. However, the limited availability, donorsite morbidity, cumbersome isolation and limited in vitro growth ofSCs restricts their use for transplantation [11,12]. To obviate such limita-tions, researchers have turned to alternative cellular sources includingmesenchymal [13–15], neural [16,17], and olfactory ensheathing stemcells [18,19] amongst other strategies [20,21] to replace lost SCs afternerve injury.

Bone marrow-derived mesenchymal stem cells are one of a fewstem cell types that have been successfully applied to the clinic for tissuerepair. In addition to theirmultipotent differentiation capacity they pos-sess a number of advantages. These include, but are not limited to, easeof isolation and maintenance, ability to survive and migrate followingtransplantation, and in permitting autologous transplantation. These to-gether can circumvent problems associated with an immune responsealong with precluding any ethical concerns. Mesenchymal stem cellsalso possess strong paracrine activity proposed as a principal mecha-nism for tissue repair and can be engineered to produce exogenoustherapeutic proteins and transdifferentiate into neural cell types [5].Such unique abilities make MSCs an invaluable cell type for neural re-pair. We posit that success with this approach would provide patientsopportunities to donate their own stem cells, which could be implantedinto nerve guidance conduits or transplanted at the site of injury inorder to facilitate nerve repair [22–25].

Mesenchymal stem cells transdifferentiation into Schwann-like cells(referred to tMSCs) and their subsequent incorporation into nerve re-generation conduits promotes axonal regeneration, reduces lesionsize, enhances neuronal survival and improves functional outcomes[14,15,26–36]. Although the success of cell transdifferentiation hasbeen assessed previously, the mechanisms underlying biochemicalandmolecular status of tMSCswith impacted pathways and proteins re-main relatively unknown.

Transdifferentiated MSCs show morphological and molecularchanges similar to native SCs. Studies have characterized this processby immunocytochemical,Western blot, enzyme-linked immunosorbentassay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR), and functional bioassays [15,24,28]. However, in prior studies,no biochemical and molecular profiles of the tMSCs were uncovered.To this end, we used proteomic and system-based pathway analysesto identify highly regulated proteins present during transdifferentiation.Such investigations uncovered pathway regulation for tMSCs into SC-like cells and the morphological and biochemical changes reflective ofthe biological outcomes. S100 expression, nerve growth factor andbrain derived neurotrophic factor (NGF and BDNF) secretion and pro-motion of neurite outgrowth were demonstrated. Proteomic analysesrevealed significant changes in protein regulation (387 out of total808) and identified pathways associated with axonal guidance,myelination, neural development and neural growth and differentia-tion. Further investigation will begin to unravel the functional state oftMSCs for neuroregeneration.

2. Materials and methods

2.1. Isolation of bone marrow-derived MSCs

Mesenchymal stem cells were isolated and cultured from BrownNorway rats (Rattus norvegicus) [24,37]. Isolated cells were propagatedin maintenance media containing alpha minimum essential media(αMEM,Gibco BRL Thermofisher Scientific,Waltham,MA, USA) supple-mented with 20% fetal bovine serum (FBS, Atlanta Biologicals, Atlanta,GA, USA), 2% 200 mM solution GlutaMAX (Gibco BRL), and 1% antibiot-ic–antimycotic (Invitrogen Thermofisher Scientific,Waltham,MA, USA)and maintained in incubators under 5% CO2 atmosphere at 37 °C. Thesub-culturing of the cells was performed every 2 to 3 days as describedin our previous work [24].

2.2. Transdifferentiation of MSCs (tMSCs) into Schwann-like cells

The protocol for in vitro, tMSCs into SC-like cells, was modified froma published report by Dezawa and colleagues [15]. The protocol waspreviously used to produce SC-like cells on micropatterned substrates[24]. Briefly, MSCs were propagated under normal growth conditionsto a confluency of 30–40% before being subjected to 1 mM β-mercaptoethanol (BME; Sigma–Aldrich, St. Louis, MO, USA) in αMEMfor one day. The following day, media from the flask was aspiratedcompletely and cells were washed with phosphate buffered saline(PBS) before subjecting them to αMEM media supplemented with10% FBS and 35 ng/ml all-trans-retinoic acid (ATRA (R2625); Sigma).After three days of incubation in retinoic acid-supplemented medium,cells were washed again with PBS and further subjected to αMEMmedia supplementedwith 20% FBS, 14 μl forskolin (FSK; EMDMillipore,Billerica, MA, USA), 5 ng/ml platelet-derived growth factor (PDGF;Sigma), 10 ng/ml basic fibroblast growth factor (bFGF, Promega Corpo-ration, Madison, WI, USA) and 200 ng/ml heregulin β1 (HRG;Calbiochem, EMDMillipore) for 8 days in vitro (DIV).

2.3. Immunocytochemistry (ICC) and ELISA tests

Transdifferentiation was assessed by examining protein expressionafter ICC using an image-based high throughput imaging system.tMSCs and undifferentiatedMSCs (uMSCs)were plated in 96well plates(655090, Greiner Bio-One, Monroe, NC, USA) at a density of 2000 cellsper well at day 12 of transdifferentiation. Cells were washed with0.1 M PO4 buffer twice and then fixed for 20 min in 4% paraformalde-hyde in 0.1 M PO4 buffer. After fixation, cells were washed with PBSthree times every 10 min and incubated in blocking solution preparedusing PBS containing 5% normal donkey serum (NDS, JacksonImmunoResearch, West Grove, PA, USA), 0.4% bovine serum albumin(BSA; Sigma) and 0.2% Triton X-100 (Fisher Scientific) for 1 h. Antibod-ies specific to glial, neuronal and proliferation markers (Table 1) werediluted to desired concentrations in blocking solution and applied tothe cells overnight at 4 °C. The following day, cells incubated with pri-mary antibodies were washed with PBS thrice every 10 min before ap-plying secondary antibodies Donkey-α-Mouse-Cy3 (1:500, JacksonImmunoResearch) or Donkey-α-Rabbit-Cy3 (1:500, JacksonImmunoResearch) alongwith DAPI (1:50) for 60–90min in the dark. Fi-nally, as the last step, cells were washed with PBS again thrice every10 min to remove any non-bound secondary antibody. 200 μl of PBSwas left in each well for fluorescence imaging. Plates were imagedusing an ImageXpress Micro high content screening system (MolecularDevices, Sunnyvale, CA, USA) and percent immunostaining for each an-tibody was quantified using the multi wavelength cell scoring moduleof the MetaXpress software (Molecular Devices, Sunnyvale, CA, USA).

A nerve growth factor-β (NGFβ) ELISA Kit (ab100757, Abcam, Cam-bridge, MA, USA) and a BDNF ELISA kit (BDNF Emax® ImmunoAssaySystem, Promega, Madison, WI, USA) were used to quantify these se-creted neurotrophic factors released from tMSCs and uMSCs. ELISAswere performed based on the instructions provided by the kit manufac-turer. Cells were plated in 6 well plates at an initial plating density of30,000 cells/well. After two days, conditioned media samples were col-lected, and cells were fixed and stained with DRAQ5 (nuclei stain)(ab108410, Abcam) to assess the number of cells present at the timeof sample collection. A Student t-test was performed to determine sig-nificant differences in the data obtained for immunocytochemistryand ELISA tests.

2.4. PC12-TrkB propagation and cell function

Genetically modified BDNF receptor expressing PC12-TrkB cells (agift fromM. Chao, New York University) were grown in RPMI-1640Me-dium (30-2001, ATCC, Manassas, VA, USA), supplemented with 10%heat-inactivated horse serum and 5% fetal bovine serum, under 5%

Table 1Antibodies used for characterization of transdifferentiated MSCs.

Primary antibody Dilution Specificity Vendor (catalog number)

Rabbit-α-S100 1:500 Glial marker (SC marker) Sigma-Aldrich (S2644)Mouse-α-S100β 1:1000 Glial marker (SC marker) Abcam (ab11178)Rabbit-α-p75NTR 1:1000 Glial marker (SC marker) Promega (G3231)Mouse-α-TUJ1 (βIII tubulin) 1:200 Neuronal marker R&D systems (MAB1195)Rabbit-α-Ki67 1:200 Proliferation marker Abcam (ab16667)

95A.D. Sharma et al. / Journal of Proteomics 165 (2017) 93–101

CO2 atmosphere at 37 °C. To test the biological activity of NGF and BDNFsecreted from tMSCs and uMSCs, neurite outgrowth of PC12-TrkB cellswas assessed in a non-contact co-culture system. Tests were performedwith tMSCs or uMSCs using transwell plateswith cell culture inserts. Forthis purpose, PC12-TrkB cells were plated onto 10 μg/ml laminin coatedwells at a density of 105 cells/well, while tMSCs or control uMSCs wereplated on top of the transwell culture inserts (CLS3450, Sigma) at a den-sity of 1.25 × 105 cells per well. Co-culture media composed of 7:3 ratioof PC12-TrkB maintenance media and MSCs maintenance media wasused to support the growth of the cell types used in the study. PC12-TrkB cells grown in co-culture media alone were used as a control.After two days in co-culture, PC12-TrkB cells were immunostainedwith βIII-tubulin antibody conjugated to Cy3 along with DAPI to stainthe nuclei. Imaging was performed using the ImageXpress Micro highcontent screening system and the average neurite extensions for eachcondition quantified using the neurite outgrowth module of theMetaXpress software. A Student t-testwas used to determine significantdifferences in PC12-TrkB neurite outgrowth data.

2.5. Proteomic assay preparation and protein analysis

The tMSCs and uMSCs were lysed [38] and proteins were quantifiedusing a Pierce 660nmProtein Assaywith 50mM ionic detergent recom-mended by the manufacturer (Thermo Fisher). 75 μg of protein weredigested usingfilter-assisted sample preparation (FASP) [39]. Followingdigestion, peptides were cleaned using Oasis MCX extraction cartridgesper manufacturer's protocol (Waters; Milford, MA). Peptides werequantified using NanoDrop2000 (Thermo Scientific; Wilmington, DE)at an absorbance of 205 nm and 2 μg of each sample was used formass spectrometry analysis. Samples were analyzed using ESI-LC-MS/MS system in a nano-spray configuration (SCIEX 6600 TripleTOF®, Fra-mingham, MA, USA) coupled with an Eksigent NanoLC 415 with acHiPLC system (Eksigent, Dublin, CA, USA). Samples were loaded ontoa 0.5 mm C18 CL 3 μm 120 Å trap column (Eksigent, Dublin, CA, USA),washed with 98:2 HPLC water with 0.1% formic acid: ACN with 0.1%formic acid for 10 min and then eluted through a 15 cm C18 CK 3 μm120 Å ChromXP column (Eksigent, Dublin, CA, USA) with 98:2 HPLCwater with 1% formic acid: ACN with 1% formic acid using a 60 min lin-ear gradient of 0–60% ACNwith 1% formic acid. The acquisition methodwas in a data-dependentmodewith one full scan followed by fragmen-tation of the 50most abundant peaks. Precursor peaks with a minimumsignal count of 100 were dynamically excluded after two selections for6 s within a range ± 25 mDa. Charge states other than 2–5 wererejected. Rolling collision energy and dynamic accumulation wereused. To create the reference spectral library for SWATH, we used fourexperimental samples and four control samples and these 8 data fileswere searched in unison using Protein Pilot software v. 5.0 (Sciex)with UniProt Swiss-Prot database (2015).

2.6. SWATH-MS performance and data independent acquisition (DIA)

For SWATH, DIA was performed using the same nanoLC conditionsas the reference spectral library. Each sample was spiked with HRM cal-ibration peptides (Biognosys AG) for retention time correction duringSWATH analysis. The SWATH acquisition method was in data-indepen-dent mode with one full scan followed by fragmentation of the 200

predetermined mass ranges, determined by a variable window calcula-tor fromAB Sciex; the smallest windowbeing 1 Da. The rangeswere de-termined based on the density of precursors at any given mass withinthe library sample. Spiked synthetic peptides were used to adjust forany changes in retention time. Rolling collision energy was used,based on a +2 charge state. Parameters for spectral alignment andtargeted data extraction using PeakView 2.2 was performed as previ-ously described [38]. The raw intensity of each proteinwas transformedusing the z-Transformation as previously described [38,40] and raw p-valueswere adjusted using Benjamini-Hochbergmultiple testingmeth-od. For each protein of interest, 2 ions for 4 peptides each were chosenfrom the spectra library performMRM (8 data points total for each pro-tein). Area under the peak was computed and used for pair-wise rela-tive quantification and the results were exported in Excel. Coefficientvariation (CV) of area counts of each ion of each conditionwas calculat-ed (stdev/avg ∗ 100) and 9 ions were removed due to poor CV's (N20%for both conditions or N30% for one condition). Next, data was importedinto Markerview 1.2.1 (Sciex) software, normalized to internal spike ofsynthetic peptide and areas under the peak for peptides were summedto protein levels for analysis and a t-test was performed.

3. Results

3.1. Expression of SCs and neuronal marker proteins in transdifferentiatedMSCs

The transdifferentiation of MSCs into Schwann-like cells (SCs) wasmonitored using an immunocytochemical analysis and indicated thattMSCs, in comparison to uMSCs, showed a greater proportion of cellsimmunolabeled for the SCs markers, S100 (33.17%), S100β (14.72%)and p75NTR (11.83%) (Fig. 1a). In addition, immunolabeling with a neu-ronal marker, TUJ1, was also greater in tMSCs (35.63%) than the uMSCs(2.91%)(Fig. 1A). The significantly elevated presence of the glial marker,S100, and the neuronal marker, TUJ1, upon transdifferentiation indi-cates the hybrid multipotential neuronal-glial fate and SCs lineage oftMSCs. Although increases of 6.7 and 2.4% in S100β and p75NTR expres-sionwere noted in tMSCs, these differenceswere not statistically signif-icant. On the other hand, a significant decrease in the proportion oftMSCs immunostained for the proliferation marker, Ki67, was noted in-dicative of the slower proliferation rate of tMSCs compared to uMSCs(Fig. 1A). The representative fluorescence images of all immunolabeledmarkers are presented in Fig. 1B. These data demonstrate that a signifi-cant proportion of the MSCs were induced toward a SC identity by thetransdifferentiation protocol.

3.2. tMSCs and uMSCs secretion of NGF and BDNF

SCs are known to secrete NGF and BDNF following injury and to ac-tivate intracellular signal transduction pathways that stimulate regen-erative axon growth and enhance neuronal survival [41–43]. Thus, wesought to determine the quantities of these neurotrophic factors secret-ed by tMSCs and the uMSCs by ELISA. The results in Fig. 2A and B showthat tMSCs secreted 968 and 271 pg/ml NGF and BDNF, respectively in24 h. These values were significantly greater than those produced byuMSCs. The increasedNGF and BDNF secreted from tMSCs is yet anotherindication of successful SC differentiation.

Fig. 1. Immunocytochemical characterization of MSC transdifferentiation. (A) Percentageimmunostaining of markers S100, S100β, p75NTR, TUJ1 and Ki67 in tMSCs and uMSCs. N= 8 and error bars represent standard error of the mean. (B) Example of pseudo-colored images of tMSCs and uMSCs immunostained for S100, βIII-tubulin, and Ki67proteins. a shows S100 (Red); a’ shows GFP (Green); a” shows DAPI (Blue) expressionin the tMSCs. a”’ shows merged image of S100, GFP, and DAPI. Similarly, b shows S100,GFP and DAPI expression in uMSCs. Rows c and d show expression of βIII-tubulin intMSCs and uMSCs respectively. Finally, rows e and f show expression of Ki67 in tMSCsand uMSCs, respectively. (Scale bar = 100 μm).


3.3. PC12-TrkB neurite outgrowth quantifications

Aneurite outgrowth assaywas developed to access the biological ac-tivity of the neurotrophic factors (NGF andBDNF) directly secreted fromtMSCs and uMSCs. In this bioassay PC12-TrkB cells were co-culturedwith either tMSCs or uMSCs under non-contact conditions usingtranswell inserts. NGF, as well as BDNF, stimulate neurite outgrowthfrom PC12-TrkB cells. PC12-TrkB cells co-cultured with tMSCs showedsignificantly longer neurites (average of 8.16 μm per cell) compared tothose co-cultured with uMSCs (average of 5.2 μm per cell) and com-pared to control PC12-TrkB cells (Fig. 3A and B). These results are inagreement with findings represented in Fig. 2A and B where thetMSCs secreted significantly higher amounts of these neurotrophic

factors. Together these results demonstrate that tMSCs (in comparisonto uMSCs) were capable of producing significantly greater amounts ofthe neurotrophic factors NGF and BDNF with potent neurite outgrowthpromoting activity.

3.4. Proteomic isolated proteins expressed in tMSCs and uMSCs

Using Panther and Ingenuity Pathway Analysis (IPA) platforms wenext investigated relevant cellular proteins by comparison of tMSCswith uMSCs. The analysis was conducted to determine differences inregulation of proteins and pathways in tMSCs (SCs) and uMSCs as a re-sult of the transdifferentiation process. The SWATH reference spectral li-brary contained 1025 proteins at the Global false discovery rate (FDR)b1% as calculated by Protein Pilot v. 5.0. In total 808 proteins were in-cluded in the SWATH analysis using PEAKView v2.2. Out of these 808,387 proteins were found to be significantly changed upontransdifferentiation (Supplementary information, Table S1). All of theproteins included in SWATH analysis (total of 808 proteins) wereuploaded to Panther classification system (http://pantherdb.org/) toclassify the proteins observed according to their biological processes(Supplementary information, Fig. S1) and protein class (Supplementaryinformation, Fig. S2). Out of these 808 proteins 32.4% are metabolic,10.6% are structural, 3.10% are immunological, 6.90% are developmental,19.50% are cellular processes, 8% are biogenesis, 8% are regulatory, 1.5%are adhesion, 0.9% are apoptotic, 4.9% are stimulus responses, 0.5% arereproduction and 3.7% are multicellular organismal process related.Three hundred and eighty-seven were differentially regulated anduploaded to the Panther database with parallel distributions. The bio-logical classes between all 808 proteins versus the 387 proteins thatare significantly regulated were compared. Biological classes thatshowed a higher proportion in the differentially regulated 387 proteinscompared to the 808 proteinswere related to immune system, develop-ment, biogenesis and apoptotic processes that show functional implica-tions to nerve regeneration. These proteins were further classifiedaccording to their biological processes and cellular components asshown in Fig. 4A and B, respectively.

A comparison of the number of upregulated (249) and downregulat-ed (138) proteins (total of 387 differentially regulated proteins) basedon their biological class was conducted and we found that proteins re-lated to metabolic processes (e.g., cellular process and cellular compo-nent organization) were significantly well-represented in the group ofupregulated proteins as compared to downregulated proteins (Fig. 5).We also tested the biological processes overrepresented in this set ofdifferentially regulated proteins, compared to all the processes presentin the organism Rattus norvegicus. Table 2 shows a comparison of bio-logical processes associatedwith this groupof 387 differentially regulat-ed proteins obtained after SWATH analysis to the biological processes ofproteins present in the organism Rattus norvegicus (total 23,781) usingPANTHER Overrepresentation Test (release 2016-07-15). Bonferronicorrection was used for multiple testing and the displayed resultswere significant (p b 0.05). This analysis identified that many proteinsinvolved with biogenesis, morphogenesis and cellular organizationwere overrepresented in the differentially regulated proteins and thisis consistent with distinct morphological differences observed betweenthe tMSCs and uMSCs.

Finally, to gain insight into which pathways were associated withthese proteins, the protein list was uploaded into the IPA program,and a list of pathways and proteins of interestwas created (Supplemen-tary information, Table S2). Pathways associated with the nervous sys-tem, cellular differentiation, proliferation, and the immune systemwere chosen as pathways of interest because of their possible involve-ment in nervous system repair (Table S3). A number of the pathwayswhich showed to be upregulated in the IPA analysis and identified bySWATH impact nerve regeneration. These include NGF, axonal guid-ance, neurotrophin/TRK and VEGF signalings.

http://pantherdb.org/

Fig. 2. tMSCs and uMSCs secrete NGFβ and BDNF. These growth factors, referred to in the figure as A and B, are released from tMSCs and uMSCs after two days of cell growth. N = 3 anderror bars represent standard error of the mean. NGFβ and BDNF secretion was significantly higher in tMSCs compared to uMSCs (p ≤ 0.05). CM refers to conditioned media.


3.5. Validation of protein expression using multiple reaction monitoring(MRM)

To validate the results from SWATH analysis, we selected seven pro-teins, which were differentially regulated and identified in pathways ofinterest. Proteins used for MRM analysis were ACTB, ACTN1, WNT5A,RTN4, GNAI2, MYL9 and ARPC2 (Table S4). Another set of 4transdifferentiation experiments were carried out (replicates 5–8) toperform the MRM analysis and to validate the SWATH results obtainedfrom replicates 1–4. To compare SWATH data of proteins found in rep-licates 1–4 versus replicates 5–8, 10 random significantly changed pro-teins were compared in the two sets and the direction of change wasfound to be the same. MRM analysis further strengthened the SWATHdata by showing similar regulation of proteins ACTB, ACTN1, WNT5A,and RTN4. Other proteins used for MRM analysis, such as GNAI2,MYL9, and ARPC2, while significantly changed in SWATH analysisgave nonsignificant results from MRM.

Fig. 3. (A) Effect of tMSCs and uMSCs on PC12-TrkB cells neurite extension using anoncontact co-culture system. N = 3 and error bars represent standard error of themean. (B) Merged immunostained images of PC12-TrkB cells using βIII-tubulin antibodyconjugated with Cy3 (Red) and DAPI (Blue) for staining nuclei. a and b show PC12-TrkBcells co-cultured (non-contact co-culture) with tMSCs and uMSCs, respectively. c and dshow negative (PC12-TrkB cells only; No soluble BDNF) and positive controls (PC12-TrkB cells + 100 ng/ml BDNF), respectively. Scale bar = 100 μm.

4. Discussion

While tMSCs are commonly used to generate SCs for neural trans-plantation strategies no prior studies have characterized the operativebiochemical and molecular transdifferentiation pathways [15,41,44,45]. Notably, common IHC, Western blot and PCR tests previously iden-tified shared tMSCS and SCs biomarkers, neurotrophic factors and func-tional outcomes for dorsal root ganglia regeneration [15,28]. Herein, weused proteomic assays to determine how transdifferentiation occurs.After transdifferentiation, differences between undifferentiated(uMSCs) and transdifferentiated MSCs (tMSCs) were assessed basedonmorphological, molecular and functional changes. Before conductingfull proteomic analysis, we conducted experiments similar to previoustransdifferentiation related research and characterized tMSCs for SCsmarker expression, neurotrophic factor secretion and functional im-provement in the form of enhanced neurite outgrowth. After consider-able in vitro characterization, we performed a detailed proteomicprofiling of both uMSCs and tMSCs to determine proteins which aredifferentially regulated. This new knowledge provides a greater under-standing of the in vitro benefits associated with the transdifferentiationof MSCs observed by us, aswell as other researchers. From a total of 808proteins selected for SWATH analysis, a large number (387) of proteinswere differentially regulated in tMSCs. In order to understand howthese differentially regulated proteins correlate with the in vitro re-search data, we looked at four important characteristics of tMSCs

(morphological andmolecular changes, secretion of growth factors, en-hanced neurite outgrowth, andmyelination) whichmay provide an ex-planation of how tMSCs provide an improvement in peripheral nerveregeneration.

Fig. 4. Classification of 387 differentially regulated proteins observed after SWATH analysis according to their protein class using Panther. (A) Classification of differentially regulatedproteins according to the biological processes they are involved in. (B) Classification of differentially regulated proteins according to their location in cellular architecture.


4.1. Morphological and molecular changes

The tested cells exhibit elongated spindle-shaped morphology withmarker confirmation of MSC transdifferentiation and demonstratedlevels of SC S100 similar to that reported previously [15,24,41,46–49].Here, transdifferentiation of bone marrow-derived MSCs to SCs was in-vestigated.We now show that the βIII-tubulin protein was upregulatedin tMSCs compared to uMSCs providing supporting evidence for neuro-nal marker expression along with SCs marker expression showing theirneuronal-glial hybrid nature.We also found that out of 387 differential-ly regulated proteins, 52 proteins were found to be related to cellularcomponent organization which may explain why tMSCs were bipolarand spindle-shaped as compared to fibroblastic tMSCs. Out of these 52differentially regulated proteins, 37 were upregulated as opposed toonly 15 proteins which were downregulated. The proteins which arefound to be responsible for regulation of cell shape were BAIP2,GDIR2, WDR1, THRB, EZRI, FINC, HEXB, MOES, MYH10, MRLCA, MYH9,PARVA, and TPM1; for regulation of cell size were EZRI, HS90A, andMOES; and for establishment and maintenance of cell polarity werePARVA, WNT5A, ARP2, ARP3, CAP1, AQP1, CDC42, COF1, and LMNA.Many of these proteins are associated with biological processes suchas actin filament organization, actin filament polymerization/

Fig. 5. Analysis of the differentially regulated proteins (387 proteins) between the tMSCswith uMSCs identified 249 upregulated and 138 downregulated proteins associated withbiological processes. Higher number of upregulated proteins for metabolic, cellular andbiogenesis associated processes were identified in tMSCs as compared to uMSCs.

depolymerization, actin filament severing, and thus helps to elucidatethe molecular mechanisms associated with the distinct bipolar mor-phology transition of the tMSCs observed in our study, as well asmany accounts in the literature. Horigane et al. [50] has shown thatWNT5A administration enhanced the axonal elongation as comparedto the dendritic outgrowth of immature cortical neurons. WNT5A sig-naling is also shown to be the multipolar-to-bipolar transition switchfor migrating neurons [51]. Similarly, another protein CDC42 whichwe observed to be differentially regulated in tMSCs, has been shownto promote Schwann cell proliferation and migration after sciaticnerve injury [52]. Another study found that COF1 (cofilin), an actindepolymerizing and severing protein, is downstream of NRG1 signalingpathway and essential for SC myelination [53].

4.2. Secretion of neurotrophic factors

Secretion of NGF and BDNF showed that tMSC production of neuro-trophic factors was unique from that of uMSCs [45] with a parallel SCphenotype. We found an increased secretion of both BDNF and NGFby tMSCs as compared to uMSCs which was found to be similar to an-other study conducted byMahay and coworkers [45]. In order to under-stand which proteins might be involved with enhanced BDNF and NGFsecretion, we looked at differentially regulated proteins involved withbiological processes associated with NGF and BDNF stimuli. We ob-served several proteins such as WNT5A, ARPC2, GRP78, RL9, RL8, RS3,and PAI1, associated with “cellular response to NGF stimulus” biologicalprocess. We also observed another differentially regulated protein, EF2,which is associated with “cellular response to BDNF stimulus” process.WNT5A, a key downstream effector of NGF, known for enhancing axo-nal branching and extension [54] was found to be upregulated intMSCs as compared to uMSCs. NGFwhich is responsible for the develop-ment of axons and dendrites by sensing cues via filopodia of growthcones was shown to recruit Arp2/3 (encoded by ARPC2) complex forstimulation of actin polymerization and formation of filopodia [55]. Inanother study, an enhanced expression of GRP78 was observed in re-lieving the endoplasmic reticulum stress-mediated apoptosis in PC12cells by activation of PI3-K/Akt signaling pathway byNGF [56]. It is note-worthy that enhanced NGF secretion by tMSCs is likely the result of dif-ferential regulation of one or several proteins working separately or inunison in tMSCs as compared to uMSCs.

4.3. Enhanced neurite outgrowth

Finally, a functional bioassay to assess neurite outgrowth promotingactivity associatedwith the tMSCswas conducted using an assay involv-ing PC12-TrkB cells. PC12-TrkB cells are known to undergo

Table 2Biological processes associatedwith differentially regulated proteins (387) obtained after SWATH analysis compared to the biological processes of proteins present in the organism Rattusnorvegicus (total 23,781) using PANTHER Overrepresentation Test (release 2016-0,7-15). Bonferroni correction was used for multiple testing and displayed results that had p b 0.05. (+)indicates overrepresentation of biological process and (−) indicates underrepresentation.Manyproteins involved in biogenesis,morphogenesis and organizationwere overrepresented inthe group of differentially regulated proteins.

PANTHER GO-slim biological process Number of proteins in Rattusnorvegicus

Number of proteins in differentially regulatedproteins (337)

Expected Foldenrichment

+/− p value

Purine nucleobase metabolic process 89 11 1.45 7.59 + 8.51E−05Protein folding 139 13 2.26 5.75 + 1.69E−04Protein complex assembly 189 16 3.08 5.2 + 3.38E−05Protein complex biogenesis 190 16 3.09 5.17 + 3.62E−05Cytokinesis 113 9 1.84 4.89 + 2.92E−02Muscle contraction 158 12 2.57 4.67 + 3.58E−03Cellular component biogenesis 681 42 11.08 3.79 + 7.39E−11Cellular component morphogenesis 537 32 8.74 3.66 + 1.31E−07Translation 693 38 11.28 3.37 + 3.05E−08Cellular component organization orbiogenesis

1972 87 32.09 2.71 + 2.77E−15

Cellular component organization 1731 68 28.17 2.41 + 3.77E−09Intracellular protein transport 1026 33 16.7 1.98 + 4.63E−02Protein metabolic process 2609 78 42.46 1.84 + 2.36E−05Biosynthetic process 1864 55 30.33 1.81 + 3.64E−03Primary metabolic process 6415 165 104.39 1.58 + 5.69E−09Metabolic process 7604 179 123.74 1.45 + 8.09E−07Cellular process 9355 189 152.24 1.24 + 2.25E−02Unclassified 9410 94 153.13 0.61 − 0.00E+00Response to stimulus 3682 28 59.92 0.47 − 1.74E−04Regulation of biological process 2930 21 47.68 0.44 − 9.94E−04Sensory perception 1602 8 26.07 0.31 − 5.45E−03Neurological system process 2100 10 34.17 0.29 − 1.27E−04


morphological differentiation by enhancing their neurite outgrowth inthe presence of both NGF and BDNF [57]. Our results showed thattMSCs stimulated greater neurite extension, supporting prior work in-vestigating tMSCs effects on regenerative neurite outgrowth from dor-sal root ganglion cells [45,46,48]. Similarly, proteomics data alsorevealed various differentially regulated proteins such as ILK, RTN4,RL24, DREB, EFNB1, MAP1B, SYUG, CSPG2, ARP3, GNAI2, THY1,CDC42, COF1, FINC, ITB1, RL4 and TRPV2, which are known to be as-sociated with nervous system development, axon guidance and axonextension. Integrin-linked kinase (ILK), which was found to be dif-ferentially regulated, is known for stimulating neurite outgrowthvia NGF [58] and also for radial sorting of axons and Schwann cellremyelination in the peripheral nervous system [59]. Similarly,RTN4 (Nogo) [60], EFNB1 [61], THY1 [62] and MAP1B [63] are alsoknown for their involvement in neural development and regenera-tion. Again, just like enhanced NGF secretion, various differentiallyregulated proteins are influencing multiple pathways and biologicalprocesses to enhance neurite outgrowth in the presence of tMSCs, asopposed to uMSCs.

4.4. Myelination

Another important property of tMSCs, as shown by a number ofstudies is their ability to myelinate axons [14,64]. To understand howtMSCs can help with myelination, we looked at the list of differentiallyregulated proteins to identify proteins (ARF6, ATPB, AT1A1, EHD3,GBB1, GBB2, NSF, NDUAA, THY1, EFTU, WDR1, ACON, ACTB, CDC42,TCPE, KCRB, CYC, ODO2, DYN1, EZRI, GELS, G6PI, AATM, HS90A,HSP7C, GRP78, CH60, HBB1, HBA, MOES, PHB, KPYM, RTN4, SEPT8,STIP1, TCPA, TKT, TBB4)whichhavepreviously been shown to be a com-ponent of the myelin sheath. In addition to finding several myelinsheath components, we also observed differential regulation of manymyelination-related gene products such as CTGF, ARF6, EZR, GELS,CAV1 and VIM. Schwann cells are known to secrete connective tissuegrowth factor (CTGF) and inhibit CNS myelination [65]. We observedthat CTGF was downregulated in tMSCs, suggesting cell-impact onmyelination. Torri and co-workers [66] proposed that Arf6 (ADP

Ribosylation Factor 6) is an intracellular signaling molecule responsiblefor mediating SC differentiation and myelination, and our SWATH anal-ysis showed that ARF6 was upregulated in tMSCs. Transforming growthfactor beta 1, an inhibitor of SC proliferation andmyelination during de-velopment [67], was found to be downregulated and is consistent withtMSCs having a greater capacity for myelination. Ezrin, which is highlyexpressed in microvilli of myelinating SC [68] was upregulated intMSCs. Actin filament severing protein gelsolin was upregulated intMSCs and is responsible for myelination of sciatic nerve after peripher-al nerve crush injury by recruitingmacrophages to the site of injury [69].The intermediate filament protein vimentin present in both SCs andneurons and known for negatively regulating myelination [70] wasfound to be downregulated in tMSCs. We also observed a significantdownregulation of Cav-1 in tMSCs, which is consistent with a decreaseof Cav-1 in the distal nerve stump after axotomy when SCs return totheir pre-myelinating phenotype [71].

Furthermore, Ingenuity Pathway Analysis (IPA) revealed pathwaysassociated with nervous and immune system regulation including axo-nal guidance signaling, NGF signaling, VEGF signaling, neuregulin sig-naling, PDGF signaling, GDNF family ligand-receptor interactions,CNTF signaling, Neurotrophin/TRK signaling, ERK/MAPK signaling,STAT3 pathway, IL-1, IL-6, IL-8, IL-22, IL-15, IL-3, TNFR1, IL-12, IL-10,IL-17, IL-9 signaling and mTOR signaling. These signaling pathways af-fect SC migration and proliferation. Some of these are operative in an-giogenesis and others in monocyte-macrophage function and areknown to affect nerve regeneration. Proteins involved in axonal guid-ance [72], VEGF signaling [73], neuregulin and PDGF [74,75] and IL-1,IL-8 and TNFR1 signaling [76,77], were significantly regulated upontMSC transdifferentiation.

In conclusion, the current study uncovered proteins and pathwaysdifferentially regulated in tMSCs. These proteins (and pathways) havesignificant implications on myelination, axonal guidance, cytokinesand growth factor secretion. This proteomic and pathway analysis hasprovided insight into how tMSCs can facilitate nerve regeneration, andthis informationmay be useful in developing strategies aimed atmanip-ulating the damaged neural tissue microenvironment to enhance nerveregeneration.


Author contributions

All of the authors made intellectual contributions to the conceptionand/or design of the study. Anup D. Sharma, Jayme Wiederin, MetinUz and Pawel Ciborowski conducted the experiments and were in-volved in data interpretation. All authors were involved in draftingand/or critical revision of the manuscript and approved the final sub-mitted version.

Disclosure of potential conflicts of interest

The authors indicate no potential conflicts of interest.

Transparency document

The Transparency document associated with this article can befound, in online version.

Acknowledgements

This researchwas funded by the US ArmyMedical Research andMa-teriel Command (grant account no.W81XWH-11-1-0700) and the StemCell Research Fund. B. Patel and Dr. E. Sandquist provided insightfulcomments to an earlier draft of this manuscript.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jprot.2017.06.011.

References

[1] J.W. Fawcett, R.J. Keynes, Peripheral nerve regeneration, Annu. Rev. Neurosci. 13(1990) 43–60.

[2] X. Navarro, M. Vivo, A. Valero-Cabre, Neural plasticity after peripheral nerve injuryand regeneration, Prog. Neurobiol. 82 (4) (2007) 163–201.

[3] U. Namgung, The role of Schwann cell-axon interaction in peripheral nerve regener-ation, Cells Tissues Organs 200 (1) (2014) 6–12.

[4] M.E. Marti, A.D. Sharma, D.S. Sakaguchi, S.K. Mallapragada, 10 - Nanomaterials forneural tissue engineering A2 - Gaharwar, A.K, in: S. Sant, M.J. Hancock, S.A.Hacking (Eds.), Nanomaterials in Tissue Engineering, Woodhead Publishing 2013,pp. 275–301.

[5] E.J. Sandquist, M. Uz, A.D. Sharma, B.B. Patel, S.K. Mallapragada, D.S. Sakaguchi, Stemcells, bioengineering, and 3-D scaffolds for nervous system repair and regeneration,in: G.L. Zhang, L.D. Kaplan (Eds.), Neural Engineering: From Advanced Biomaterialsto 3D Fabrication Techniques, Springer International Publishing, Cham 2016,pp. 25–81.

[6] M. Uz, A.D. Sharma, P. Adhikari, D.S. Sakaguchi, S.K. Mallapragada, Development ofmultifunctional films for peripheral nerve regeneration, Acta Biomater.(2016)http://dx.doi.org/10.1016/j.actbio.2016.09.039.

[7] S.K. Mallapragada, T.M. Brenza, J.M. McMillan, B. Narasimhan, D.S. Sakaguchi, A.D.Sharma, S. Zbarska, H.E. Gendelman, Enabling nanomaterial, nanofabrication andcellular technologies for nanoneuromedicines, Nanomedicine 11 (3) (2015)715–729.

[8] T. Hadlock, C. Sundback, D. Hunter, M. Cheney, J.P. Vacanti, A polymer foam conduitseeded with Schwann cells promotes guided peripheral nerve regeneration, TissueEng. 6 (2) (2000) 119–127.

[9] G.R. Evans, K. Brandt, S. Katz, P. Chauvin, L. Otto, M. Bogle, B.Wang, R.K.Meszlenyi, L.Lu, A.G. Mikos, C.W. Patrick Jr., Bioactive poly(L-lactic acid) conduits seeded withSchwann cells for peripheral nerve regeneration, Biomaterials 23 (3) (2002)841–848.

[10] G.E. Rutkowski, C.A. Miller, S. Jeftinija, S.K. Mallapragada, Synergistic effects ofmicropatterned biodegradable conduits and Schwann cells on sciatic nerve regener-ation, J. Neural Eng. 1 (3) (2004) 151–157.

[11] C. Mauritz, C. Grothe, K. Haastert, Comparative study of cell culture and purificationmethods to obtain highly enriched cultures of proliferating adult rat Schwann cells,J. Neurosci. Res. 77 (3) (2004) 453–461.

[12] T.K. Morrissey, N. Kleitman, R.P. Bunge, Isolation and functional characterization ofSchwann cells derived from adult peripheral nerve, J. Neurosci. Off. J. Soc. Neurosci.11 (8) (1991) 2433–2442.

[13] C.J. Chen, Y.C. Ou, S.L. Liao, W.Y. Chen, S.Y. Chen, C.W. Wu, C.C. Wang, W.Y. Wang,Y.S. Huang, S.H. Hsu, Transplantation of bone marrow stromal cells for peripheralnerve repair, Exp. Neurol. 204 (1) (2007) 443–453.

[14] T. Mimura, M. Dezawa, H. Kanno, H. Sawada, I. Yamamoto, Peripheral nerve regen-eration by transplantation of bone marrow stromal cell-derived Schwann cells inadult rats, J. Neurosurg. 101 (5) (2004) 806–812.

[15] M. Dezawa, I. Takahashi, M. Esaki, M. Takano, H. Sawada, Sciatic nerve regenerationin rats induced by transplantation of in vitro differentiated bone-marrow stromalcells, Eur. J. Neurosci. 14 (11) (2001) 1771–1776.

[16] T. Murakami, Y. Fujimoto, Y. Yasunaga, O. Ishida, N. Tanaka, Y. Ikuta, M. Ochi,Transplanted neuronal progenitor cells in a peripheral nerve gap promote nerve re-pair, Brain Res. 974 (1–2) (2003) 17–24.

[17] W. Heine, K. Conant, J.W. Griffin, A. Hoke, Transplanted neural stem cells promoteaxonal regeneration through chronically denervated peripheral nerves, Exp. Neurol.189 (2) (2004) 231–240.

[18] E. Verdu, X. Navarro, G. Gudino-Cabrera, F.J. Rodriguez, D. Ceballos, A. Valero, M.Nieto-Sampedro, Olfactory bulb ensheathing cells enhance peripheral nerve regen-eration, Neuroreport 10 (5) (1999) 1097–1101.

[19] C. Radtke, A.A. Aizer, S.K. Agulian, K.L. Lankford, P.M. Vogt, J.D. Kocsis, Transplanta-tion of olfactory ensheathing cells enhances peripheral nerve regeneration after mi-crosurgical nerve repair, Brain Res. 1254 (2009) 10–17.

[20] W. Martens, K. Sanen, M. Georgiou, T. Struys, A. Bronckaers, M. Ameloot, J. Phillips, I.Lambrichts, Human dental pulp stem cells can differentiate into Schwann cells andpromote and guide neurite outgrowth in an aligned tissue-engineered collagen con-struct in vitro, FASEB J. 28 (4) (2014) 1634–1643.

[21] L. Ziegler, S. Grigoryan, I.H. Yang, N.V. Thakor, R.S. Goldstein, Efficient generation ofSchwann cells from human embryonic stem cell-derived neurospheres, Stem CellRev. Rep. 7 (2) (2011) 394–403.

[22] H. Klingemann, D. Matzilevich, J. Marchand, Mesenchymal stem cells - sources andclinical applications, Transfus. Med. Hemother. 35 (4) (2008) 272–277.

[23] A.D. Sharma, P.A. Brodskiy, E.M. Petersen, M. Dagdeviren, E.A. Ye, S.K. Mallapragada, D.Sakaguchi, High throughput characterization of adult stem cells engineered for deliv-ery of therapeutic factors for neuroprotective strategies, Jove-J Vis Exp. 95 (2015).

[24] A.D. Sharma, S. Zbarska, E.M. Petersen, M.E. Marti, S.K. Mallapragada, D.S. Sakaguchi,Oriented growth and transdifferentiation of mesenchymal stem cells towards aSchwann cell fate on micropatterned substrates, J. Biosci. Bioeng. 121 (3) (2016)325–335.

[25] S. Wakao, D. Matsuse, M. Dezawa, Mesenchymal stem cells as a source of Schwanncells: their anticipated use in peripheral nerve regeneration, Cells Tissues Organs200 (1) (2014) 31–41.

[26] M.M. Harper, S.D. Grozdanic, B. Blits, M.H. Kuehn, D. Zamzow, J.E. Buss, R.H. Kardon,D.S. Sakaguchi, Transplantation of BDNF-secreting mesenchymal stem cells providesneuroprotection in chronically hypertensive rat eyes, Invest. Ophthalmol. Vis. Sci. 52(7) (2011) 4506–4515.

[27] T.V. Johnson, N.D. Bull, D.P. Hunt, N. Marina, S.I. Tomarev, K.R. Martin, Neuroprotec-tive effects of intravitreal mesenchymal stem cell transplantation in experimentalglaucoma, Invest. Ophthalmol. Vis. Sci. 51 (4) (2010) 2051–2059.

[28] G. Keilhoff, A. Goihl, K. Langnase, H. Fansa, G. Wolf, Transdifferentiation of mesen-chymal stem cells into Schwann cell-like myelinating cells, Eur. J. Cell Biol. 85 (1)(2006) 11–24.

[29] J.D. Kocsis, Neuroprotection and immunomodulation by cell transplantation are be-coming central themes in potential therapeutic approaches for cell-based therapies,Neurosci. Lett. 456 (3) (2009) 99.

[30] K. Kurozumi, K. Nakamura, T. Tamiya, Y. Kawano, K. Ishii, M. Kobune, S. Hirai, H.Uchida, K. Sasaki, Y. Ito, K. Kato, O. Honmou, K. Houkin, I. Date, H. Hamada, Mesen-chymal stem cells that produce neurotrophic factors reduce ischemic damage in therat middle cerebral artery occlusion model, Mol. Ther. 11 (1) (2005) 96–104.

[31] Y. Li, M. Chopp, Marrow stromal cell transplantation in stroke and traumatic braininjury, Neurosci. Lett. 456 (3) (2009) 120–123.

[32] M. Sasaki, C. Radtke, A.M. Tan, P. Zhao, H. Hamada, K. Houkin, O. Honmou, J.D.Kocsis, BDNF-hypersecreting human mesenchymal stem cells promote functionalrecovery, axonal sprouting, and protection of corticospinal neurons after spinalcord injury, J. Neurosci. Off. J. Soc. Neurosci. 29 (47) (2009) 14932–14941.

[33] L.R. Zhao, W.M. Duan, M. Reyes, C.D. Keene, C.M. Verfaillie, W.C. Low, Human bonemarrow stem cells exhibit neural phenotypes and ameliorate neurological deficitsafter grafting into the ischemic brain of rats, Exp. Neurol. 174 (1) (2002) 11–20.

[34] W. Zheng, O. Honmou, K. Miyata, K. Harada, J. Suzuki, H. Liu, K. Houkin, H. Hamada,J.D. Kocsis, Therapeutic benefits of human mesenchymal stem cells derived frombone marrow after global cerebral ischemia, Brain Res. 1310 (2010) 8–16.

[35] S.R. Das, M. Uz, S. Ding, M.T. Lentner, J.A. Hondred, A.A. Cargill, D.S. Sakaguchi, S.Mallapragada, J.C. Claussen, Electrical differentiation of mesenchymal stem cellsinto Schwann-cell-like phenotypes using inkjet-printed graphene circuits, Adv.Healthc. Mater. 6 (2017) 1601087.

[36] M. Uz, M. Buyukoz, A.D. Sharma, D.S. Sakaguchi, S.A. Altinkaya, S.K. Mallapragada,Gelatin-based 3D conduits for transdifferentiation of mesenchymal stem cells intoSchwann cell-like phenotypes, Acta Biomater. 53 (2017) 293–306.

[37] E.-A. Ye, S.S. Chawla, M.Z. Khan, D.S. Sakaguchi, Bonemarrow-derivedmesenchymalstem cells (MSCs) stimulate neurite outgrowth from differentiating adult hippo-campal progenitor cells, Stem Cell Biol. Res. 3 (1) (2016) 3.

[38] N.A. Haverland, H.S. Fox, P. Ciborowski, Quantitative proteomics by SWATH-MS re-veals altered expression of nucleic acid binding and regulatory proteins in HIV-1-in-fected macrophages, J. Proteome Res. 13 (4) (2014) 2109–2119.

[39] J.R. Wisniewski, A. Zougman, N. Nagaraj, M. Mann, Universal sample preparationmethod for proteome analysis, Nat. Methods 6 (5) (2009) 359–362.

[40] C. Cheadle, M.P. Vawter, W.J. Freed, K.G. Becker, Analysis of microarray data using Zscore transformation, J. Mol. Diagn. JMD 5 (2) (2003) 73–81.

[41] G. Keilhoff, A. Goihl, F. Stang, G. Wolf, H. Fansa, Peripheral nerve tissue engineering:autologous Schwann cells vs. transdifferentiated mesenchymal stem cells, TissueEng. 12 (6) (2006) 1451–1465.

[42] B. Schlosshauer, E. Müller, B. Schröder, H. Planck, H.-W. Müller, Rat Schwann cells inbioresorbable nerve guides to promote and accelerate axonal regeneration, BrainRes. 963 (1–2) (2003) 321–326.

http://dx.doi.org/10.1016/j.jprot.2017.06.011doi:10.1016/j.jprot.2017.06.011doi:10.1016/j.jprot.2017.06.011http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0005http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0005http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0010http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0010http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0015http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0015http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0020http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0020http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0020http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0020http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0025http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0025http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0025http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0025http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0025http://dx.doi.org/10.1016/j.actbio.2016.09.039http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0035http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0035http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0035http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0035http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0040http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0040http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0040http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0045http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0045http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0045http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0045http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0045http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0045http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0050http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0050http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0050http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0055http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0055http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0055http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0060http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0060http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0060http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0065http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0065http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0065http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0070http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0070http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0070http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0075http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0075http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0075http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0080http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0080http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0080http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0085http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0085http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0085http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0090http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0090http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0090http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0095http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0095http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0095http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0100http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0100http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0100http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0100http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0105http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0105http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0105http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0110http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0110http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0115http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0115http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0115http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0120http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0120http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0120http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0120http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0125http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0125http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0125http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0130http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0130http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0130http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0130http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0135http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0135http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0135http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0140http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0140http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0140http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0145http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0145http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0145http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0150http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0150http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0150http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0150http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0155http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0155http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0160http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0160http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0160http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0160http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0165http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0165http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0165http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0170http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0170http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0170http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0175http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0175http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0175http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0175http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0180http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0180http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0180http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0185http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0185http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0185http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0190http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0190http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0190http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0195http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0195http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0200http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0200http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0205http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0205http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0205http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0210http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0210http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0210


[43] C.A. Krekoski, D. Neubauer, J. Zuo, D. Muir, Axonal regeneration into acellular nervegrafts is enhanced by degradation of chondroitin sulfate proteoglycan, J. Neurosci.Off. J. Soc. Neurosci. 21 (16) (2001) 6206–6213.

[44] D. Mahay, G. Terenghi, S.G. Shawcross, Growth factors in mesenchymal stem cellsfollowing glial-cell differentiation, Biotechnol. Appl. Biochem. 51 (Pt 4) (2008)167–176.

[45] D. Mahay, G. Terenghi, S.G. Shawcross, Schwann cell mediated trophic effects by dif-ferentiated mesenchymal stem cells, Exp. Cell Res. 314 (14) (2008) 2692–2701.

[46] J. Caddick, P.J. Kingham, N.J. Gardiner, M. Wiberg, G. Terenghi, Phenotypic and func-tional characteristics of mesenchymal stem cells differentiated along a Schwann celllineage, Glia 54 (8) (2006) 840–849.

[47] G. Keilhoff, F. Stang, A. Goihl, G. Wolf, H. Fansa, Transdifferentiated mesenchymalstem cells as alternative therapy in supporting nerve regeneration and myelination,Cell. Mol. Neurobiol. 26 (7–8) (2006) 1235–1252.

[48] P.J. Kingham, D.F. Kalbermatten, D. Mahay, S.J. Armstrong, M. Wiberg, G. Terenghi,Adipose-derived stem cells differentiate into a Schwann cell phenotype and pro-mote neurite outgrowth in vitro, Exp. Neurol. 207 (2) (2007) 267–274.

[49] M. Tohill, C. Mantovani, M. Wiberg, G. Terenghi, Rat bone marrow mesenchymalstem cells express glial markers and stimulate nerve regeneration, Neurosci. Lett.362 (3) (2004) 200–203.

[50] S. Horigane, N. Ageta-Ishihara, S. Kamijo, H. Fujii, M. Okamura, M. Kinoshita, S.Takemoto-Kimura, H. Bito, Facilitation of axon outgrowth via a Wnt5a-CaMKK-CaMKIalpha pathway during neuronal polarization, Mol. Brain 9 (2016) 8.

[51] M. Boitard, R. Bocchi, K. Egervari, V. Petrenko, B. Viale, S. Gremaud, E. Zgraggen, P.Salmon, J.Z. Kiss, Wnt signaling regulates multipolar-to-bipolar transition of migrat-ing neurons in the cerebral cortex, Cell Rep. 10 (8) (2015) 1349–1361.

[52] B. Han, J.Y. Zhao, W.T. Wang, Z.W. Li, A.P. He, X.Y. Song, Cdc42 promotes Schwanncell proliferation and migration through Wnt/beta-catenin and p38 MAPK signalingpathway after sciatic nerve injury, Neurochem. Res. 42 (5) (2017) 1317–1324.

[53] N. Sparrow, M.E. Manetti, M. Bott, T. Fabianac, A. Petrilli, M.L. Bates, M.B. Bunge, S.Lambert, C. Fernandez-Valle, The actin-severing protein cofilin is downstream ofneuregulin signaling and is essential for Schwann cell myelination, J. Neurosci. 32(15) (2012) 5284.

[54] D. Bodmer, S. Levine-Wilkinson, A. Richmond, S. Hirsh, R. Kuruvilla,Wnt5amediatesnerve growth factor-dependent axonal branching and growth in developing sympa-thetic neurons, J. Neurosci. Off. J. Soc. Neurosci. 29 (23) (2009) 7569–7581.

[55] D.J. Goldberg, M.S. Foley, D. Tang, P.W. Grabham, Recruitment of the Arp2/3 com-plex and mena for the stimulation of actin polymerization in growth cones bynerve growth factor, J. Neurosci. Res. 60 (4) (2000) 458–467.

[56] S. Kishi, K. Shimoke, Y. Nakatani, T. Shimada, N. Okumura, K. Nagai, K. Shin-Ya, T.Ikeuchi, Nerve growth factor attenuates 2-deoxy-d-glucose-triggered endoplasmicreticulum stress-mediated apoptosis via enhanced expression of GRP78, Neurosci.Res. 66 (1) (2010) 14–21.

[57] Y. Iwasaki, M. Ishikawa, N. Okada, S. Koizumi, Induction of a distinct morphologyand signal transduction in TrkB/PC12 cells by nerve growth factor and brain-derivedneurotrophic factor, J. Neurochem. 68 (3) (1997) 927–934.

[58] J. Mills, M. Digicaylioglu, A.T. Legg, C.E. Young, S.S. Young, A.M. Barr, L. Fletcher, T.P.O'Connor, S. Dedhar, Role of integrin-linked kinase in nerve growth factor-stimulat-ed neurite outgrowth, J. Neurosci. Off. J. Soc. Neurosci. 23 (5) (2003) 1638–1648.

[59] J.A. Pereira, Y. Benninger, R. Baumann, A.F. Gonçalves, M. Özçelik, T. Thurnherr, N.Tricaud, D. Meijer, R. Fässler, U. Suter, J.B. Relvas, Integrin-linked kinase is requiredfor radial sorting of axons and Schwann cell remyelination in the peripheral nervoussystem, J. Cell Biol. 185 (1) (2009) 147.

[60] J.L. Goldberg, B.A. Barres, Nogo in nerve regeneration, Nature 403 (6768) (2000)369–370.

[61] S.M. Shah, Y.J. Kang, B.L. Christensen, A.S. Feng, R. Kollmar, Expression of Wnt recep-tors in adult spiral ganglion neurons: frizzled 9 localization at growth cones ofregenerating neurites, Neuroscience 164 (2) (2009) 478–487.

[62] C.H. Chen, S.M.Wang, S.H. Yang, C.J. Jeng, Role of Thy-1 in in vivo and in vitro neuraldevelopment and regeneration of dorsal root ganglionic neurons, J. Cell. Biochem. 94(4) (2005) 684–694.

[63] P.R. Gordon-Weeks, I. Fischer, MAP1B expression and microtubule stability in grow-ing and regenerating axons, Microsc. Res. Tech. 48 (2) (2000) 63–74.

[64] G. Keilhoff, F. Stang, A. Goihl, G. Wolf, H. Fansa, Transdifferentiated mesenchymalstem cells as alternative therapy in supporting nerve regeneration and myelination,Cell. Mol. Neurobiol. 26 (7) (2006) 1233–1250.

[65] R. Lamond, S.C. Barnett, Schwann cells but not olfactory ensheathing cells inhibitCNS myelination via the secretion of connective tissue growth factor, J. Neurosci.33 (47) (2013) 18686–18697.

[66] T. Torii, Y. Miyamoto, M. Yamamoto, K. Ohbuchi, H. Tsumura, K. Kawahara, A.Tanoue, H. Sakagami, J. Yamauchi, Arf6 mediates Schwann cell differentiation andmyelination, Biochem. Biophys. Res. Commun. 465 (3) (2015) 450–457.

[67] S. Einheber, M.J. Hannocks, C.N. Metz, D.B. Rifkin, J.L. Salzer, Transforming growthfactor-beta 1 regulates axon/Schwann cell interactions, J. Cell Biol. 129 (2) (1995)443–458.

[68] S.S. Scherer, T. Xu, P. Crino, E.J. Arroyo, D.H. Gutmann, Ezrin, radixin, and moesin arecomponents of Schwann cell microvilli, J. Neurosci. Res. 65 (2) (2001) 150–164.

[69] A.F. Gonçalves, N.G. Dias, M. Moransard, R. Correia, J.A. Pereira, W. Witke, U. Suter,J.B. Relvas, Gelsolin is required for macrophage recruitment during remyelinationof the peripheral nervous system, Glia 58 (6) (2010) 706–715.

[70] D. Triolo, G. Dina, C. Taveggia, I. Vaccari, E. Porrello, C. Rivellini, T. Domi, R. La Marca,F. Cerri, A. Bolino, A. Quattrini, S.C. Previtali, Vimentin regulates peripheral nervemyelination, Development 139 (7) (2012) 1359–1367.

[71] D.D. Mikol, S.S. Scherer, S.J. Duckett, H.L. Hong, E.L. Feldman, Schwann cell caveolin-1 expression increases during myelination and decreases after axotomy, Glia 38 (3)(2002) 191–199.

[72] M. Nozumi, T. Togano, K. Takahashi-Niki, J. Lu, A. Honda, M. Taoka, T. Shinkawa, H.Koga, K. Takeuchi, T. Isobe, M. Igarashi, Identification of functional marker proteinsin the mammalian growth cone, Proc. Natl. Acad. Sci. U. S. A. 106 (40) (2009)17211–17216.

[73] J.M. Rosenstein, J.M. Krum, C. Ruhrberg, VEGF in the nervous system, Organ 6 (2)(2010) 107–114.

[74] N. Baker, G. Zhang, Y. You, R.S. Tuan, Caveolin-1 regulates proliferation and osteo-genic differentiation of human mesenchymal stem cells, J. Cell. Biochem. 113 (12)(2012) 3773–3787.

[75] S. Parrinello, I. Napoli, S. Ribeiro, P.W. Digby, M. Fedorova, D.B. Parkinson, R.D.S.Doddrell, M. Nakayama, R.H. Adams, A.C. Lloyd, EphB signaling directs peripheralnerve regeneration through Sox2-dependent Schwann cell sorting, Cell 143 (1)(2010) 145–155.

[76] P.M. Vidal, E. Lemmens, D. Dooley, S. Hendrix, The role of “anti-inflammatory” cyto-kines in axon regeneration, Cytokine Growth Factor Rev. 24 (1) (2013) 1–12.

[77] P. Dubovy, R. Jancalek, T. Kubek, Role of inflammation and cytokines in peripheralnerve regeneration, Int. Rev. Neurobiol. 108 (2013) 173–206.

http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0215http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0215http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0215http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0220http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0220http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0220http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0225http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0225http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0230http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0230http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0230http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0235http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0235http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0235http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0240http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0240http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0240http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0245http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0245http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0245http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0250http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0250http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0250http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0255http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0255http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0255http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0260http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0260http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0260http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0265http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0265http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0265http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0265http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0270http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0270http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0270http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0275http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0275http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0275http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0280http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0280http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0280http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0280http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0285http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0285http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0285http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0290http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0290http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0290http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0295http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0295http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0295http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0295http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0300http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0300http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0305http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0305http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0305http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0310http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0310http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0310http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0315http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0315http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0320http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0320http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0320http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0325http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0325http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0325http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0330http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0330http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0330http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0335http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0335http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0335http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0340http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0340http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0345http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0345http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0345http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0350http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0350http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0350http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0355http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0355http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0355http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0360http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0360http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0360http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0360http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0365http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0365http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0370http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0370http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0370http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0375http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0375http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0375http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0375http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0380http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0380http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0385http://refhub.elsevier.com/S1874-3919(17)30221-X/rf0385

Proteomic analysis of mesenchymal to Schwann cell transdifferentiation1. Introduction2. Materials and methods2.1. Isolation of bone marrow-derived MSCs2.2. Transdifferentiation of MSCs (tMSCs) into Schwann-like cells2.3. Immunocytochemistry (ICC) and ELISA tests2.4. PC12-TrkB propagation and cell function2.5. Proteomic assay preparation and protein analysis2.6. SWATH-MS performance and data independent acquisition (DIA)

3. Results3.1. Expression of SCs and neuronal marker proteins in transdifferentiated MSCs3.2. tMSCs and uMSCs secretion of NGF and BDNF3.3. PC12-TrkB neurite outgrowth quantifications3.4. Proteomic isolated proteins expressed in tMSCs and uMSCs3.5. Validation of protein expression using multiple reaction monitoring (MRM)

4. Discussion4.1. Morphological and molecular changes4.2. Secretion of neurotrophic factors4.3. Enhanced neurite outgrowth4.4. Myelination

Author contributionsDisclosure of potential conflicts of interestTransparency documentAcknowledgementsAppendix A. Supplementary dataReferences

journal of proteomics · journal of proteomics 165 (2017) 93–101 ⁎...

Documents