Expression of dopamine-associated genes on conjunctiva stromal-derived human mesenchymal stem cells

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Biochemical and Biophysical Research Communications 377 (2008) 423–428

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Biochemical and Biophysical Research Communications j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y b b r c

Expression of dopamine-associated genes on conjunctiva stromal-derived human mesenchymal stem cells Samad Nadri a,b,c, Masoud Soleimani d,*, Zahra Mobarra e, Sepideh Amini a a

Stem Cells and Tis­sue Engi­neer­ing Depart­ment, Stem Cell Tech­nol­ogy Insti­tute, Teh­ran, Iran Med­i­cal Phys­ics and Bio­med­i­cal Engi­neer­ing Depart­ment, Fac­ulty of Med­ic­ ine, Sha­hid Be­he­shti Uni­ver­sity of Med­i­cal Sci­ence, Teh­ran, Iran c Nano­tech­nol­ogy Depart­ment, Stem Cell Tech­nol­ogy Insti­tute, Teh­ran, Iran d Hema­tol­ogy Depart­ment, Fac­ulty of Med­i­cal Sci­ence, Tarb­i­at Mod­ares Uni­ver­sity, Teh­ran 14115-111, Iran e Molec­u­lar Biol­ogy Depart­ment, Stem Cell Tech­nol­ogy Insti­tute, Teh­ran, Iran b

a r t i c l e

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Article history: Received 23 September 2008 Available online 11 October 2008  Key­words: Con­junc­tiva mes­en­chy­mal stem cells Dopa­mine neu­ron Par­kin­son’s dis­eases

a b s t r a c t Par­kin­son’s dis­ease (PD) is neu­ro­de­gen­er­a­tive dis­eases caused by the loss of dopa­mi­ner­gic neu­rons in the sub­stan­tia nigra pars com­pacta. Stem cell ther­apy is one of the prom­is­ing strat­e­gies in help­ing to cure PD. In the pres­ent study, human mes­en­chy­mal stem cells (MSCs) from eye con­junc­tiva stro­mal cells were dif­fer­en­ti­ated into dopa­mi­ner­gic neu­rons. In this work, after con­junc­tiva biopsy, mes­en­chy­mal stem cells were obtained via adher­ence to the plas­tic cul­ture dishes. Then, MSCs were treated with gen­eral neu­ro­genic medium con­tain­ing DMEM sup­ple­ mented with RA, IBMX and dbcAMP for 6 days. RT-PCR, immu­no­cy­to­chem­is­try and flow cytom­e­try were used for expres­sion of dopa­mi­ner­gic genes such as TH. As a result, RT-PCR anal­y­sis revealed the expres­sion of dopa­mi­ner­gic neu­ron genes such as TH, Ptx3, Nurr1. Fur­ther­more, immu­no­cy­to­chem­is­try revealed that the dif­fer­en­ti­ated CJMSCs not only express TH gene, but also express TH pro­tein. Flow cytom­e­try showed that TH, MAP-2 pro­teins increased sig­nif­i­ cantly as increas­ing pas­sage num­ber. In con­clu­sion, the reported results indi­cate that CJMSCs might be a suit­able and avail­able source for cell trans­plan­ta­tion ther­apy for the cen­tral sys­tem dis­eases such as PD. © 2008 Else­vier Inc. All rights reserved.

Par­kin­son’s dis­ease (PD) is a com­mon neu­ro­de­gen­er­a­tive dis­or­ der of the brain which has as a part of its core pathol­ogy the pro­ gres­sive degen­er­at­ ion of the dopa­mi­ner­gic nigro­stri­a­tal path­way. It affects all eth­nic groups at a fre­quency of »1% of people >65 years of age. Although sev­eral treat­ments have been shown to mod­ify the course of the dis­ease, none have suc­cess­fully halted the degen­er­at­ ion [1,2]. In recent years, stem cell replace­ment has emerged as the novel ther­ap ­ eu­tic strat­egy for Par­kin­son’s dis­ease (PD). Stem cells are defined as a pop­u­la­tion of prim­i­tive cells with the capa­bil­ity of self-renewal and dif­fer­en­ti­a­tion into multiple cell lin­ eages [3]. These cells have been iso­lated from a wide vari­ety of tis­sue. There are many reports on the der­i­va­tion of dopa­mine neu­rons from human embry­onic stem cells [4–9]. How­ever, ESCs can form ter­ato­ mas and are likely to elicit immune rejec­tion if used for trans­plan­ta­ tion. Neu­ral stem cells (NSCs) main­tain line­age spec­i­fic­ity and have the abil­ity to dif­fer­en­ti­ate into any type of brain cell [10]. Although NSCs show ther­a­peu­tic value for neu­ral dis­or­ders, NSC-derived dopa­ mine neu­rons have some draw­backs with respect to effi­ciency and line­age polar­i­za­tion [11]. This by no means sug­gests that NSCs and ESCs will not tran­si­tion from bench to bed­side, but addi­tional stud­ies * Cor­re­spond­ing author. Fax: +98 21 88970478. E-mail addresses: so­leim_m@mod­ares.ac.ir (S. Masoud).solei­man­i_ma­soud@ yahoo.com (S. Masoud). 0006-291X/$ - see front matter © 2008 Else­vier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.09.148

are war­ranted to iden­tify other stem cells with the abil­ity to gen­er­ate dopa­mine neu­rons with high effi­ciency. The least char­ac­ter­ized stem cells, with respect to their abil­ity to gen­er­ate dopa­mine neu­rons, are the mes­en­chy­mal stem cells. Of the adult stem cells, MSCs appear to have the best potential for regen­er­a­tive med­i­cine [12–17]. In fact, MSCs are cur­rently in clin­i­cal tri­als for a num­ber of dis­or­ders includ­ ing graft ver­sus host dis­ease, heart fail­ure, multiple scle­ro­sis [17] and Par­kin­son­ism [18]. Mes­en­chy­mal stem cells from the con­junc­ti­val stroma of the human eye pos­ses stem cells prop­er­ties. We pre­vi­ously dem­on­ strated that con­junc­ti­val stroma-derived MSCs (CJMSCs) could be induced to dif­fer­en­ti­ate into neu­ron-like cells. CJMSCs are also capa­ble of dif­fer­en­ti­at­ing into oste­o­genic, chon­dro­genic and adi­ po­genic [19]. In the pres­ent study, CJMSCs were iso­lated and trans­formed into dopa­mi­ner­gic neu­rons in vitro. Stud­ies like this could help rec­og­nize a new source of MSCs for treat­ment of PD. Mate­ri­als and meth­ods Iso­la­tion, expan­sion and char­ac­ter­iza­tion of CJMSCs. CJMSCs were iso­lated accord­ing to a pro­to­col mod­i­fied from Nadri and co-worker [19]. In brief, After informed con­sent from the ­indi­vid­u­als, 2–3 mm2

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con­junc­tiva biopsy was obtained from patients ­under­go­ing ­ptery­gium sur­gery and it was incu­bated in sup­ple­mented hor­ monal epi­the­lial medium (SHEM), which is made of Dul­becco’s mod­i­fied Eagle’s medium (DMEM)/F-12 (GIB­CO-BRL, Grand Island, NY) con­tain­ing bicar­bon­ate (Sigma Chem­i­cal Co., St. Louis, MO), 0.5% dimethyl sulf­ox­ide, 2 ng/ml human epi­der­mal growth fac­tor (EGF; Pep­ro­tech, Rocky Hill, NJ), 5 mg/ml insu­lin (GIB­COBRL), 5 mg/ ml trans­fer­rin (GIB­CO-BRL), 5 ng/ml sodium sel­e­nite (GIB­CO-BRL), 0.5 mg/ml hydro­cor­ti­sone (GIB­CO-BRL), 30 ng/ml chol­era toxin A sub­unit, 5% fetal calf serum (FCS; GIB­CO-BRL), 50 mg/ml gen­ta­mi­ cin (Sigma Chem­i­cal Co.), and 1.25 mg/ml ampho­ter­i­cin B (Sigma Chem­i­cal Co.) for 2–5 min. Then, the biop­sies were treated in SHEM con­tain­ing 50 mg/ml dis­pase II (Sigma Chem­i­cal Co.) and 100 mM sor­bi­tol (Es­pan­a et al., 2003). Under a ste­reo­mi­cro­scope, epi­the­ lial sheets were sep­a­rated and the iso­lated stro­mal tis­sue seg­ment was cul­tured in DMEM/F-12 (1:1) (GIB­CO-BRL) sup­ple­mented with 10% knock­out serum (GIB­CO-BRL), 4 ng/ml basic-FGF (Pep­ro­ tech), 5 mg/ml insu­lin (Sigma Chem­i­cal Co.), and 10 ng/ml human LIF (Chem­icon; Teme­cu­la, CA) (Drav­ida et al., 2005) and incu­bated at 37 °C with 5% CO2 in a humid­ifi ­ ed cham­ber for 2 weeks. After 2 weeks, the biopsy was removed and the mes­en­chy­mal stem cells were tryp­sin­i­zed (0.25% Tryp­sin–eth­yl­ene­di­amine­tet­ra­ace­tic acid [EDTA]; GIB­CO-BRL) after con­flu­ency. The cells were sub­se­quently expanded by two pas­sages in DMEM (GIB­CO-BRL) sup­ple­mented with 100 IU/ml pen­i­cil­lin (Sigma Chem­i­cal Co.), 100 mg/ml strep­ to­my­cin (Sigma Chem­i­cal Co.), and 15% FCS. To iden­tify MSCs nature of the iso­lated cells, the cells were treated with oste­o­genic DMEM com­posed of 50 mg/ml ascor­bic acid 2-phos­phate (Sigma Chem­i­cal Co.), 10 nM dexa­meth­a­sone (Sigma Chem­i­cal Co.), and 10 mM b-glyc­er­op ­ hos­phate (Sigma Chem­i­cal Co.), adi­po­genic (DMEM, sup­ple­mented with 50 mg/ml in­do­meth­a­cine (Sigma Chem­i­cal Co.) and 100 nM dexa­meth­a­sone (Sigma Chem­i­cal Co.); and chon­dro­genic DMEM sup­ple­mented with 10 ng/ml trans­form­ing growth fac­tor-ß3 (TGF-ß3; Sigma Chem­i­cal Co.), bone mor­pho­ge­netic pro­tein-6 (BMP-6), 10¡7 M dexa­meth­a­sone (Sigma Chem­ic­ al Co.), and 50 mg/ml ascor­bate2-phos­phate (Sigma Chem­i­cal Co.), 50 mg/ml insu­lin–trans­fer­rin– sele­nium (ITS; GIB­CO-BRL) medium for 21 days [19]. For cell sur­face marker (anti­gen) char­ac­ter­iza­tion, MSCs mark­ ers were mea­sured by flow cytom­e­try using FITC-con­ju­gated anti-human CD34 (Milt­en ­ yi Bio­tec GmbH, Berg­isch Glad­bach, Ger­ many), CD73 (Ab­gent, San Diego, CA), CD105, CD45 (eBio­science; San Diego, CA), and PE-con­ju­gated anti-human CD44, Al­cam (CD166) (eBio­science). In vitro dif­fer­en­ti­a­tion of cells to dopa­mi­ner­gic neu­ron. The cells (CJMSCs), hav­ing been thawed and plated at 100 cell/cm2 in a flask 25 cm2, were incu­bated in DMEM sup­ple­mented with 100 IU/ml pen­i­cil­lin (Sigma Chem­i­cal Co.), 100 mg/ml strep­to­my­ cin (Sigma Chem­i­cal Co.), and 15% FCS for 4 days until con­flu­ency was achieved. The pro­lif­er­a­tion medium was replaced with a neu­ ro­genic medium con­sist­ing of DMEM, sup­ple­mented with 0.5 mM iso­bu­tyl methyl xan­thin (IBMX; Sigma Chem­ic­ al Co.), 1 mM di­bu­ ty­ryl-cAMP (dbcAMP; Sigma Chem­ic­ al Co.), and 10 mM ret­i­noic acid (Sigma Chem­i­cal Co.) [19] for 6 days. At the end of this period, the cells were used for RT-PCR anal­y­sis and immu­no­cy­to­chem­is­try. Two, sixth and ten-pas­sage cells were used for the exper­i­ments includ­ing RT-PCR for dopa­mi­ner­gic gene expres­sion, immu­no­cy­ to­chem­is­try and flow cytom­e­try anal­y­sis for MAP-2 and TH pro­ teins. RT-PCR anal­y­sis. Total RNA was iso­lated from cells by using the Nu­cle­o­spin RNAII kit (Mache­rey-Na­gel, Ger­many). Prior to reverse tran­scrip­tion (RT), RNA sam­ples were digested with DNa­seI (EN0521; Fer­men­tas) to remove con­tam­i­nat­ing geno­ mic DNA. DNase I was dis­solved in 10£ reac­tion buffer with MgCl2, and 1 U/ll of DNase I was added per 1 lg of RNA and incu­bated for 30 min at 37 °C. DNa­seI activ­ity was arrested fol­

low­ing addi­tion of 1 ll of 25 mM EDTA and it was incu­bated at 65 °C for 10 min. Stan­dard RT was per­formed using the Re­vert­ Aid™ H Minus First Strand cDNA Syn­the­sis Kit (Fer­men­tas) and 2 lg total RNA, 0.5 lg oligo (dt18) per reac­tion accord­ing to the man­u­fac­turer’s instruc­tions. Reac­tion mix­tures for PCR included 2.5 ll cDNA, 1£ PCR buffer (AMS™, Sin­agen, Iran), 200 lM dNTPs, 0.5 lM of each of For­ward and Reverse prim­ers and 1 U Taq DNA poly­mer­ase(Fer­men­tas, MD, USA). The prim­ers are listed in Table 1. Poly­mer­ase chain reac­tions were per­formed at 94 °C for 1 min, 25–30 cycles 94 °C for 30 s, 55–63 °C for 30 s, and 72 °C for 45 s and 72 °C for 7 min. Ampli­fied DNA frag­ments were elec­tro­pho­re­sed on 1.5% aga­rose gel. The gels were stained with ethi­dium bro­mide (10 lg/ml) and pho­to­graphed on a UV trans­il­lu­mi­na­tor (uvi­doc, UK). Immu­no­cy­to­chem­is­try anal­y­sis. The cells treated with neu­ro­ genic medium were cul­tured on ster­ile glass cover slips and fixed by incu­ba­tion in 1% para­for­mal­de­hyde/PBS for 3–5 min, per­me­ abi­li­zed with 0.5% Tri­ton X-100 in PBS for 15 min, and post-fixed for an addi­tional 10 min in 4% para­for­mal­de­hyde in PBS. The cells were then reacted with primary anti­bod­ies (mouse anti-tyro­sine hydrox­y­lase (TH), and Micro­tu­bule-asso­ci­ated pro­tein-2 (MAP-2; Chem­icon) at 4 °C for 24 h, washed with PBS and reacted with the iso­thi­oc­ y­a­nate (FITC)-con­ju­gated anti mouse IgG as the sec­ond­ary anti­body (Sigma Chem­i­cal Co.) at room tem­per­a­ture for 3 h. Then, the cells were washed with PBS-Tween 0.1% three times and incu­ bated with di­am­inobenzi­dine (DAB) solu­tion (Sigma Chem­i­cal Co.) for 10 min. Flow cytom­e­try anal­y­sis. The cul­ti­vated cells (pas­sage 2, 6, 10) in neu­ro­genic medium were detached with Tryp­sin/EDTA and per­ me­abi­li­zed with 0.5% Tri­ton X-100 in PBS for 45 min. About 2 £ 105 cells were divided into ali­quots in amber-tinted 5 ml cen­tri­fuge tubes and 3% rat serum was added. The cells were incu­bated on ice for 30 min, resus­pended in 400 ll PBS and pel­leted by cen­tri­fu­ga­ tion for 10 min at 400g. Next, the cells were stained with fluo­res­ cent iso­thi­o­cy­a­nate (FITC)-con­ju­gated mouse anti-human MAP-2 (Chem­icon) and TH (CE­DAR­LANE) at a con­cen­tra­tion of 2 lg/ml at

Table 1 Spe­cific prim­ers used for PCR ampli­fi­ca­tion. Genes

Primer sequences

Size (bp)

EN1

CTGGGTGTACTGCACACGT­TAT TACT­CGCTCTCGTCTTTGTCCT

356

EN2

GTGGGTCTACTGTACGCGCT CCTACTCGCTGTCCGACTTG

358

Nurr1

GCACTTCGGCAGAGTTGAATGA GGTGGCTGTGTTGCTGGTAGTT

491

Ptx3

TGGGAGTCTGCCTGTTG­CAG CAG­CGAACCGTCCTCTGGG

213

Pax2

AT­GTTCGCCTGGGAGATTCG GCAAGTGCTTCCGCAA­ACTG

361

Wnt1

TAG­CCTCCTCCACGAACCTG CAG­CCTCGGTTGACGATCTTG

239

Wnt3a

AAGCAGGCTCTGGGCAGCTA GAC­GGTGGTGCAGTTCCA

234

Wnt5a

AT­CCTGACCACTGGAAGCCCTGT GGCTCATGGCGTT­CAC­CAC

358

TH

GTCCCCTGGTTCCCAAGAAAAGT TCCAGCTGGGGGATATTGTCTTC

333

HPRT1

CCTGGCGTCGTGATTAGTG TCAGTCCTGTCCATAATTAGTCC

125



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4 °C for 3 h. The cells were also stained with FITC-labeled mouse anti-rat IgG 1, 2 (Sigma) as neg­at­ ive con­trols. The cells were pel­ leted, washed twice with PBS and fixed with 1% para­for­mal­de­hyde in PBS. After fix­a­tion, FACS anal­y­sis was per­formed on a FACS Cal­ li­bur cytom­e­try (Bec­ton Dick­in­son, San Jose, CA) using cell quest soft­ware. Win MDI 2.8 soft­ware was used to cre­ate the his­to­ grams. Results Cell cul­ture and char­ac­ter­iza­tion of CJMSCs In this work, the fibro­blast-like cells from human con­junc­tiva stro­mal cells (n = 10) were iso­lated by adhe­sion of these cells on the sur­face of the plas­tic cul­ture dishes. After 2 weeks, fibro­blast-like cells with spindle-shape mor­phol­ogy appeared on cul­ture dishes. To con­firm mes­en­chy­mal nature, the fibro­blast-like cells were treated with appro­pri­ate osteo-, chon­dro- and adipo-induc­tive media (Nadri et al., 2008), and their dif­fer­en­ti­a­tion was con­firmed via appro­pri­ate stain­ing includ­ing aliz­a­rin red (for oste­o­genic dif­ fer­en­ti­a­tion), alcian blue (for chon­dro­genic dif­fer­en­ti­at­ ion) and oil red (for adi­po­genic dif­fer­en­ti­a­tion) stain­ing (Sup­ple­men­tary File 1). Fur­ther­more, flow cytom­e­try anal­y­sis showed that the expres­ sion cell sur­face mark­ers such as SH2, SH3, CD44, CD166 (Al­cam) was positive, but CD34 and CD45 was neg­at­ ive (Sup­ple­men­tary File 2). In these cells, spindle-shaped mor­phol­ogy with pro­lif­er­a­tion and dif­fer­en­ti­at­ ion capac­ity was main­tained dur­ing the subculture period and up to pas­sage 15. Dopa­mi­ner­gic dif­fer­en­ti­at­ ion of the cells Next, we stud­ied the potential of CJMSCs to dif­fer­en­ti­ate into dopa­mi­ner­gic neu­ral cells. To elu­ci­date the dopa­mi­ner­gic neu­ral dif­fer­en­ti­a­tion potential of CJMSCs, these cells were cul­tured in neu­ro­in­duc­tive medium includ­ing DMEM sup­ple­mented with RA, IBMX and dbcAMP. After 6 days, we exam­ined the expres­ sion of dopa­mi­ner­gic genes by RT-PCR and immu­no­cy­to­chem­ is­try. RT-PCR anal­y­sis was indic­at­ ive of the expres­sion of TH and Nurr1 genes in treated com­pared with non-treated cells (Fig. 1). Immu­no­cy­to­chem­is­try was used to ana­lyze the intra­ cel­lu­lar TH and MAP-2 pro­teins. These pro­teins were detected in the CJFLCs after treat­ment in neu­ro­in­duc­tive medium for 6 days (Fig. 2). Flow cytom­e­try anal­y­sis The treated cells (pas­sages 2, 6 and 10) in neu­ro­genic medium were ana­lyzed for the expres­sion of a MAP-2 and TH pro­teins, as shown in Fig. 3. Results showed that the expres­sion of MAP-2 and TH pro­teins cells appears to grad­u­ally increase as the num­ber of pas­sages rises. About 95% of the cells (pas­sage 10) expressed TH and MAP-2 pro­teins and did not lose dif­fer­en­ti­at­ ion potential up to pas­sage 15 (Data not shown). Dis­cus­sion Par­kin­son dis­ease is the sec­ond most com­mon neu­ro­de­gen­er­a­ tive dis­or­der. The lifetime risk of devel­op­ing Par­kin­son dis­ease for men and women is 2% and 1.3%, respec­tively. A range of dif­fer­ent option is cur­rently used to reverse the symp­toms of PD. Patients ini­tially respond to treat­ment with dopa­mi­ner­gic-enhanc­ing med­ i­ca­tions such as levo­dopa [20,21]. How­ever, the effec­tive­ness of such treat­ments grad­u­ally dimin­ishes because the con­ver­sion to dopa­mine within the brain is increas­ingly dis­rupted by the pro­ gres­sive degen­er­a­tion of the dopa­mi­ner­gic ter­mi­nals. As a result,

Fig. 1. Inves­ti­ga­tion of dopa­mi­ner­gic-spe­cific gene expres­sion. Expres­sion of dopa­ mi­ner­gic mark­ers in con­trol (day 0) and dif­fer­en­ti­a­tion cul­ture (day 6). HPRT is shown as a con­trol for RNA sam­ple qual­ity.

most patients with Par­kin­son’s dis­ease suf­fer from dis­abil­ity that can­not be sat­is­fac­to­rily con­trolled [21]. An alter­na­tive approach that has been tar­geted using for the ren­o­va­tion of the dam­aged dopa­mi­ner­gic sys­tem is appli­ca­tion of stem cells [22]. To date, sev­eral dif­fer­ent stem cells have been inves­ti­gated for PD dis­eases, such as embry­onic stem cells (ESCs) [6,23,24] and mes­en­chy­mal stem cells. How­ever, dif­fi­cul­ties of using human ESCs includ­ing the eth­i­cal issues and tumor­i­genic potential led to the search for other types of cells for treat­ment of PD dis­ease. These include the pos­si­bil­ity of autol­o­gous grafts using MSCs, as well as allo­graft of human ESCs. The dif­fer­en­ti­a­tion capa­bil­ity of MSCs into dopa­mi­ner­gic neu­ ron has been described [25]. Despite the fact that the bone mar­row is con­sid­ered to be a well-accepted source of MSCs, the clin­i­cal use of MSCs from this source has pre­sented prob­lems includ­ing pain­ful aspi­ra­tion, con­tam­i­na­tion with non-MSCs, the low yield of aspi­ra­ tion and declin­ing stem cells prop­er­ties with increas­ing pas­sage num­ber [26]. This has led many research­ers to inves­ti­gate alter­ nate sources of MSCs for treat­ment of PD dis­ease. To date, these cells have been dif­fer­en­ti­ated into dopa­mi­ner­gic neu­rons from Whar­ton’s Jelly [27] tis­sues. This pro­ject aimed to induce CJMSCs into mature dopa­mine secret­ing cells.

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Fig. 2. Immu­no­stain­ing of con­junc­tiva mes­en­chy­mal stem cells with TH and MAP-2 stain­ing. The cells were main­tained in neu­ro­genic medium for 6 days and ana­lyzed for expres­sion of TH and MAP-2 pro­teins (A, C). Cells were co-stained with 4,6-di­ami­di­no-2-phen­yl­in­dole to visu­al­ize nuclei (blue) related to TH (B, D). (For inter­pre­ta­tion of the ref­er­ences to color in this fig­ure leg­end, the reader is referred to the web ver­sion of this paper.)

Fig. 3. Expres­sion of TH and MAP-2 pro­teins dur­ing diverse pas­sages. The cells were main­tained in neu­ro­genic medium for 6 days and ana­lyzed for expres­sion of TH and MAP-2 pro­teins in two, sixth and tenth pas­sages.



S. Nadri et al. / Biochemical and Biophysical Research Communications 377 (2008) 423–428

Con­junc­tiva tis­sue has been iden­ti­fied as an alter­na­tive source of mul­ti­po­tent stro­mal MSCs, which can be obtained by a less inva­ sive method with­out any con­tam­i­na­tion with non-MSCs, and it can main­tain stem cell capa­bil­ity includ­ing pro­lif­er­a­tion and osteoadipo-chon­dro­genic dif­fer­en­ti­a­tion up to pas­sages 15. The cells iso­lated (fibro­blast-like cells) from con­junc­tiva stro­mal tis­sue, along with their capa­bil­ity in dif­fer­en­ti­at­ing into bone, adi­po­cyte and chon­dro­cyte lin­eages and expres­sion of cell sur­face anti­gens such as CD105 (SH2), CD73(SH3), CD166 (Al­cam) con­vinced us that the cells being exam­ined are the mes­en­chy­mal stem cells. In this work, CJMSCs expressed dopa­mi­ner­gic neu­ron genes after treat­ment with DMEM con­tain­ing 10% FBS, 0.5 mM IBMX, 1 mM dbcAMP, and 10 mM ret­i­noic acid for 1 week. Pre­vi­ous inves­ ti­ga­tors have dif­fer­en­ti­ated MSCs in 2 weeks using a mul­ti­step pro­to­col in cock­tail of dopa­mi­ner­gic medium con­sist­ing of DMEM sup­ple­mented with 10% FBS, sonic hedge­hog (SHH), fibro­blast growth fac­tor, epi­der­mal growth fac­tor, N2 sup­ple­ment, butyl­ated hydroxy­an­i­sole, IBMX, RA and dbcAMP [25,27]. Accord­ing to results it seems that CJMSCs dif­fer­en­ti­ated into dopa­mi­ner­gic neu­rons by an eas­ier pro­to­col com­pared with BMMSCs and human umbil­i­cal mes­en­chy­mal stem cells. In the pres­ent study, RT-PCR anal­y­sis was used for the con­ fir­ma­tion of dif­fer­en­ti­at­ ion of CJMSCs into dopa­mi­ner­gic neu­ rons. RT-PCR anal­y­sis showed that dopa­mi­ner­gic genes includ­ing TH, En1, En2, PTX3, Wnt1, Wnt3a and Wnt5a were expressed in CJMSCs. Fur­ther­more, immu­no­cy­to­chem­is­try and flow cytom­e­try anal­y­sis revealed that TH pro­tein was expressed in CJMSCs and the per­cent­age of it had been increased by sub­cul­tures. Sev­eral stud­ies of dopa­mi­ner­gic induc­tion reported TH expres­sion in MSCs after induced dif­fer­en­ti­a­tion [28–30]. A TH pro­tein is the ini­tial and rate-lim­it­ing enzyme in the cat­e­chol­amine syn­the­sis path­way, and is con­sid­ered the prin­ci­pal reg­u­la­tor of dopa­mine bio­syn­the­sis in the cen­tral neu­ron sys­tem. In this work, sur­pris­ingly the expres­ sion rate of TH and MAP-2 pro­teins were grad­u­ally increased as a num­ber of pas­sages rises which indi­cated CJMSCs are pro­gen­ i­tor cells that increased neu­ro­genic dif­fer­en­ti­at­ ion as ris­ing pas­ sage num­ber. How­ever, com­pared to bone mar­row-derived MSCs, CJMSCs are preserve neu­ro­genic dif­fer­en­ti­at­ ion capac­ity up to high pas­sages. Prin­ci­ple donor cells for Par­kin­son’s dis­ease ther­apy should be eas­ily avail­able, capa­ble of rapid expan­sion in cul­ture, long-term sur­vival, and long-term expres­sion of genes such as TH [31]. The results in this work showed that CJMSCs were obtained via a sim­ple approach, main­tained rapid pro­lif­er­at­ ion capac­ity and expressed TH gene and pro­tein up to 15 pas­sages. Fur­ther­more, the expres­ sion and detec­tion of TH and some other dopa­mine-asso­ci­ated genes such as Nurr1, PTX3, En1, En2, Wnt1, Wnt3a nec­es­sary for both the sur­vival and dif­fer­en­ti­a­tion of the mes­en­ce­phalic dopa­ mi­ner­gic pre­cur­sor’s neu­rons [32–36] showed us that CJMSCs are dif­fer­en­ti­ated into dopa­mi­ner­gic neu­rons and are good can­di­dates for neu­ro­log­ic­ al cell ther­apy. Col­lec­tively, results of our study indi­cated that CJMSCs expressed dopa­mi­ner­gic genes using a sim­ple method. This high potential of dopa­mi­ner­gic dif­fer­en­ti­at­ ion together with high pro­lif­er­a­tion and long-term cul­ture of cells allowed us to con­clude that these cells are prom­is­ing can­di­dates for treat­ing Par­kin­son’s dis­ease. Acknowl­edg­ment This work was sup­ported by a grant from Stem Cells Tech­nol­ogy insti­tute. Appen­dix A. Sup­ple­men­tary data Sup­ple­men­tary data asso­ci­ated with this arti­cle can be found, in the online ver­sion, at doi:10.1016/j.bbrc.2008.09.148.

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