Nghiên cứu biệt hóa tế bào gốc trung mô máu cuống rốn người thành tê bào thần kinh, tế bào cơ tim và tế bào tiết insulin bằng dịch chiết mô

Tap chi Cong nghe Sinh hoc 6(4): 415-422, 2008 NGHIEN CITU BIT HOA TE BAO GOC TRUNG MO MAU CUONG RON NGU6I THANH TE BAO THAN KINH, TE BAO CO TIM VA TE BAO TIET INSULIN BANG BICH CHIET MO Phum Van Pluic, Nguyen Bang Khoa, Triin Sao Kiem, Phan Kim Ngoc Trufmg DO hoc Khoa hoc Ty' nhien, Ali hoc Quac gia pha Ha Chi Minh TOM TAT Te bao gee co the biet hem dual tic Ong cita nhieu tic nhan khic nhu hia chit, tic nhan vot chat nen, chuyen gen, deng nuoi cay va dich chi& met Trong do, Web chi& me dirge sir clang tucmg doi phO bien; khong chi de chimg minh kha nang biet heta to bao goc ma nhrtm chimg minh kha nang biet h6a dm no kieu hinh cia mo ton tgi vi mot throng dm mo Trong nghien ciru nay, cac to bao gee trung me tir mau cutmg 'On nguth dime thin nghiem Wet h6a to bao than kinh, ca tim va to bao tier insulin bang (itch chiet tir nao thai chueit, tim heo va chwjt Te bao goc trung mo fir miu cueng ron dime thu nh'i'n va chirng minh theo cac quy trinh truerc day da cong bo Kieu hinh to bao sau biet hint duqc xac dinh dva vao hinh clang d.c trung, nhuOm 116a me va sv bleu hien gen chuyen biet thong qua Ic'y thuit RT-PCR Ket qui cho thay, cac to bao gOc trung me M man cueng rein co kha nang biet hem kieu hinh to bao cita me thu Bich chiet moi trtrong be sung Web chiet ty le thich hop CS neng de 400 µg/ml protein dich chiet thu tir chuOt, to bao gee biet h6a cic cum dao vOi hieu qua cao nhat cac Wing ldiao sat Cic cvm dao Neu hien cac gen chuyen biet cho nhu Glut-2, Ngn3, Pax4 va Insulin C mot truing b8 sung 10% dich chiet nao, sv biet h6a cixa to bao goc cic neurosphere xay manh; clic to bao tir neurosphere bieu hien duang tinh v&i nestin Ci moi truiffng be sung 1% dich chiet tim, to bao gee trung mo thay del hinh clang gieng hinh dans to bao ca tim va bieuhien gen cTnT dic cho to bao ca tim NhAng ket qua cho thay, (Itch chiet mO ci the lam thay doi kieu hinh to bao getc theo kieu With cia mo dich Tit khea: Biet hoa, cuang ran nguai, dich chiet ma, to biro ca tim, to bao goc, to biro than kinh MO DAU Te bao gee ten tai o hAu het cic me cita ca the tnrong Chong c6 vai fro quan trong viec tri can bang WM mo Tat ca cac m8 co the duqc cau thank tir cac to bao cochirc nang.pe trinh bt tic dOng ter to bio c6 chirc nang, nhirng to bao gee ton tai mei luon o cic vi tri chien lucre niOt khoing khong gian rieng biet, gvi la (niche) to bio goc la vi moi truerng gilt!) cho to bao gee ton tai tv lam moi - khong biet h6a Khi khong gian ben thieu, to bio gee chui khoi 6; to day vOi cic tic dOng to bao dA c6 chirc,nang se chin biet hint kieu hinh Dieu d6 gqi y rang cic yeu to cia mo c6 tic dOng leo lii sv Wet h6a cua to bao gOc (Collas et al., 2006; Hakelien et al., 2006) Te bao goc tir mo ma ngueri c6 the biet hew to bao co kieu hinh giOng bio tiet insulin dual tic Ong cia dich chiet (Lee et al., 2008), to bao ca tim dueri tic Ong cia dich chiet ca tim (Gaustad et al., 2004; Schimrosczyk et al., 2008) re bao gee trung mo tir thy xuang chuOt cimg duqc chimg minh la oft kha nang biet h6a to bio tier insulin duai tic Ong cia dich chi& (Choi et al., 2005) Nguyen bio sqi tir thai chuOt el-mg c6 the cam irng biet hew bao tiet insulin nuoi moi triremg be sung dich chi& to bao ung thu tiet insulin tuyen (Hakelien et al., 2006), hay nguyen bao sch 293T cimg ceo the bi Net hee to bao lympho T neu duqc cam img bii dich chiet to bao T (Hakelien et al., 2002) Dich chi& xuang bo duqc chimg minh c6 nang gay biet h6a to bao goc trung mo nguai to bao xuang (Woo et al., 2006) Trong nghien cim dm Liu va (long We gii (2005) cho thay, dich chi& tir nga rang c6 the cam 1mg biet hew cac bao hen than nhi rang cac bao giOng nguyen bao nga rang (Liu et al., 2005) Cac to bio goc phoi cling c6 the duqc biet hew dyed tic Ong cha dich chiet to bao cita me dich nhu to bao quin (Qin et al., 2005) Tren ca so cac ketxqua cia cac nghien ciru hen day, nghien ciru tiep tic sir dwig dich chiet de 415 PhamVanPhilc et al kiem tra kha,nang biet hoa cUa bao gac,trung mo mau cuong rein ngtrai thinh to bao gi8ng to bao than kinh bang dich chiet nao thai chuet, to bio ca tim bang dich chiet tim heo vi to bao fiat insulin bang dich chiet toy chuet chua truang VAT LIEU VA PHLTdNG PHAP Thu nhan to bito gOc trung ma mau men ran ngtrai (hUCB MSC) Mau cuong rein thu nhan tir cac sin da duqc xet nghiem am tinh y6i HIV, HBV, HCV o Benh vien Himg Vuang pha He Chi Minh Thu nhan quan the bao don nhan bang phucmg phap ly tam ten gradient Ficoll-paque (Sigma) it toc a 2500 yang/phut, phin MSC duqc Lich khoi to bio gec too mau van the re bao dam nhan bang phuang phap nuoi cay va mai truang chon loc Te bio dam nhan sau thu nhan duqc huyen phu moi truing nuoi cay IMDM (Sigma), 10% FBS (Invitrogen, Gibco), birth nuoi cay (25 cm', Nunc) cho dot mat a 3.105 bito/cm2 o dieu kien 37°C, 5% CO2 Sau 48 h, cac MSC bit da'u barn ten be mat birth nuoi, thay men twang de looi be cac to bao khong barn (cac to bao chat va bao giic to mau), tiep toc nuoi den to bio dot mat a 70 80% dien tich be mat binh nuoi cay vii the de thay moi truing la ngay/lan Cfic to bio thu /than ter Ian cay chuyen thir se duqc sir dong Wet h6a (Phan Kim Ngoc et al., 2007; 2008) Tyo (Itch chiet tyy Toy chu'Ut sau thu nhan duqc cat nhuyen dung dich PBS ba sung 10% FBS Ly tam 3000 vong/phut 10 phut, thu nhan dich noi cho eppendorf 1,5 ml sach Tiep tic ly tam 12000 yong/phin 20 phut, thu ratan dich nen Dich noi duqc sir ding de b8 sung vao moi truing biet h6a vii L-DMEM (Sigma), a cac nZing protein tang so cila dich chiet la 100, 200, 300, 400, 500 va 600 µg/ml Vo tang dich chiet bang cich WC qua ming loc 0,2 gm Moi truing co ba sung dich chi& t duqc tit lonh toi da sau pha Tao dich chiet nao Nao thai chuet 18 dpc duqc tach, u lonh va nghien bang cod, ding nhat nao dung dich PBS va ethanol de dot the tich la 1,5 ml Dich nao dong nhat duqc dong lonh qua dem nitrogen long, sau do, giai dong chain bang cach u it 4°C Sau tan di, dich nao duqc ly tam a 3000 yang/phut phin 416 va thu dich noi Dich noi age ba sung yeti huyet theo tk le 1:1; sau do, loc bang ming loc 0,2 gm va bao quan o —20°C cho sir dong wan Tyo dich chiet ca tim heo Tim heo tir - thing tu6i duqc can de xac dinh klufn lucmg N6 duqc chuyen vao dia chira sin dung dich PBS/Gentamycin va cat nho Sau do, ly tam him hqp ma' ca tim vita cat de loot be PBS, AM them ml trypsin 0,25%/100 mg m8, khuay mau Ito bang 'ca khuay tiro toc a 200 yang/phut, 30 phtit, 36,5°C Sau do, thu dich noi, ly tam 3000 yang/phut 10 phut de thu can bio, tai huyen phi" can bang ml moi truong, at it 4°C Te bao ca tim duqc phi ye bang song sieu am & buem song trung birth theo chu 16, phat xung 30 giay, ngimg 30 giay; lien toc 30 phut Sau ly giai, dich d8ng nhat duqc ly tam 12.000 vong/phut 15 phut, it 4°C va thu dich noi Dich noi duqc dong lath nitrogen long qua am; sau giai dong nhanh va loc qua ming 0,2 gm Sir dong tuan Cam ung bit hoa Wing dich chiet hUCB MSC dang phat trien phi' kin chitng 70% be mat dong co nuoi duqc thay men tramg biet hem tuang img cho vies cam img biet h6a to bao tiet insulin, to bao co tim va bao than kinh la moi truing nuoi be sung dich chiet tuyen toy, mei ca tim va mei nao Men twang biet hem duqc thay vii the da ngay/lan MCA tnr&ng biet h6a hUCB MSC to bio tiet insulin: L-DMEM (Sigma), be sung 100, 200, 300, 400, 500 va 600 µg/ml protein dich chiet tong so M6i tnrang biet hem hUCB MSC to bao giang to bao than, kinh: IMDM (Sigma) 2% FBS (Gibco), bit sung lin lugt 1, 5, 10 va 15% dich chiet nao thai chuet theo quy trinh ten Med twang biet has hUCB MSC bao ca tim: IMDM (Sigma) 2% FBS (Gibco) ba sung 0,5, I, va 10% dich chiet Te bao sau biet h6a tir to bito MSC duqc xac dinh thong qua so thay den hinh dong, nhuem h6a mei va RT-PCR vii cac marker ac Nhuam hoa ma Nhuam dithizone (Sigma): binh nuoi to bao sau cam img biet hem duqc be moi truing, rira hai Ian bang co dinh bang ethanol tuyet 15 phut Sau do, ink to bao duqc it thuac nhuOm dithizone 30 phut it 37°C; bo thuac Tgp chi eonk frighe Sinh hpc 6(4): 415-422, 2008 binePBS 'ita quay sat so hinh nhuOm; rira lai phirc hqp maikki trong'cac to bao tiet insulin va cum to bio giang tuY dad Phan img RT-PCR RNA tang se &roc tach bang Trizol (SigmaAldrich) theo huong clan cua nha san xuat dinh Wong bang may quang phe UV/vis De chay PCR, lig RNA tong so duqc chuyen cDNA sir dung SuperScript II reverse transcriptase (Promega) v6i oligo (dT) (Promega,' USA) va random hexamer primers (Promega) PCR duqc tien hanh sir dung Taq DNA polymerase (Promega) Tat ca phin img PCR duqc tien hanh sir dung may lua'n nhiet PCR (EppendorO San pham duqc phan tich dien di ten gel agarose 3% Ten va trinh to cac primer, kich thy& cua san phim PCR, chu Icy nen hanh va nhiet de lai cho mai cap mai duqc het ke bang Gen 13-actin sir dung nhu met doi chimg Mai (internal control) Bang Cac primer sir dung nghien ciru Gene Size (bp) Insulin 263 Ngn3 313 Pax4 496 Glut-2 298 Beta actin 587 Nestin 495 cTnT 250 Sequence GCAGCCTTTGTGAACCAACA GTTGCAGTAGTTCTCCAGGTG GGTAGAAAGGATGACGCCTC CCGAGTTGAGGTCGTGCAT AGGAGGACCAGGGACTACCGT TTTAGGTGGGGTGTCACTCAG GTACAATGACAGAAGATAAG TGCTACTAACATGGCTTTGA CCAAGGCCAACCGCGAGAAGATGAC AGGGTACATGGTGGTGCCGCCAGAC AGAGGGGAATTCCTGGAG CTGAGGACCAGGACTCTCTA OGCAGCGGAAGAGGATGCTGAA GAGGCACCAAGTTGGGCATGAACGA KET QUA VA THAO LUAN Bitt h6a to bio hUCB MSC to ban tit insulin hail htt cite to thi nghie'm v6i cac ming dO djch chiet lchac nhau, cac to bio hUCB MSC den thay d8i hinh clang sau thay mai truang Wet h6a 24 h M8i trirang biet hea v6i 100, 200, 500 va 600 ug/m1 djch chi& lam to bao bong len be mat dung cu nuoi va chet nhieu sau 24 va 48 h nuoi Trong d6, tnrong biet h6a veri nOng d6 protein djch chi& tong so la 300, 400 µg/ml, se to bao chet it Mn di, so thay doi hinh dang xay chair, nhieu to bao co lai moi va tap hop nhieu cum giong nhu toy dao vao 10 (Hinh 2A, B) So phan img cua btlo be sung dich chi& co5 Annealing Cycles temperature 45 30 45 30 45 30 43 30 60 30 53 35 60 35 the nhieu ca the Ichac Met se tac gia cho rang co the so phan img cua to bio 1i bat lqi djch chiet lam thay dei ip suat thauSdo phan protein b8 sung vao); mat so cho rang de la phan img cac thank phan djch chiet (chit protein) gay tac Ong Met se gia thiet cho rang rang c6 the phan DNA djch chiet tac Ong len so bieu hien gen to bio Nhung ca the cu the cua van de chua duqc chimg minh r8 rang; c6 le phu thuec vao timg twang hqp Trong nghien ciru nay, so phan ung cua to bao bo sung dich chiet vai protein thap (100 vi 200 µg/ml) va cao (500, 600 µg/ml) lam cho bac) bong len va chet nhieu Wang to nhu 18 dei chung khong be sung djch chiet (mai tnrang L-DMEM) Wei tnrong L-DMEM , moi twang ngheo dinh clueing duqc sir dung de gay stress cho so biet h6a 417 Phan Van Ph& et al Cac to bit) hUCB MSC hau nhu khong the song va phat trien,ducm m8i truang khong be, sung huyet 0, m8i truing bo sung 100, 200 ug/m1 protein ton so djch chiet co the chua dap Ong dti Minh phan chat can duet de tri su phat then cua cac hUCB MSC nen to bao chet Trong d6, mai truing be sung 500, 600 µg/ml thi phan protein qua cao lam thay dOi ap suit them thau va to bao ding chet Trong do, hai ming di) 300 va 400 ug/m1 protein djch chiet, thi s8 luting to bao chet rat it, hau het cac to bio tiep t lc phat teen va thay doi hinh clang theo th6i gian Trong nghien dm nay, day la hai ming d8 thick hop that cac neng d8 tren deu co su xuat hien cac clan VC*, bao giong tny dao va dieu khans xay 18 doi chimg IChi nhuem voi dithizone, tat ca cac clue to bio giong tny dao deu bat man thuoc nhueim va nhieu to bao don ding bat mau Khi dem s8 lucmg clue to bao va to bio ducrng tinh thuoc nhuOm dithizone cho they m8i truang biet h6a voi 400 tg/m1 protein tong so tit dich chiet thi hieu qua biet hoa cao that voi 13,3 gun to bao ducmg tinh thuoc nhuOm/thi truing (Hinh 1A) Khi kiem tra RT-PCR 618 biet h6a be sung 400 ug/m1 protein djch chiet, cho they to bao bieu hien Glut-2, Ngn3, Pax4 vi Insulin (Hinh 1B) 14 ■ 12 10 100 200 300 A 400 500 600 Ladder Beta actin Control Glut-2 Ngn3 Control Pax4 Control Insulin B Hinh (A) Riau qua biet Ma cac ntong dO dich chiet khac (5' ding de 400 pg/ml, hiOu qui biet hoa xay cao nhat Va nong dO 100 hay 600 pg/ml thi than nh6t (B) Ket qua chay RT-PCR xac djnh Si,, bieu hion mot se gen chuyen biOt cho to bac) tint insulin Kat qua cho they, to bao sau biet hoa 'Au hien cac gen quan cho to bao tiet insulin nhu, Glut-2, Ngn3, Pax4 va Insulin Bitt hos hUCB MSC thinh to bar) giOng to ban than kinh KM cam Ong biC't hem vai mei tnrang be sung djch chiet, so phin Ong cue to bio vii moi tnrong co khic cac neng de djch chiet ,khfic Nhan xet chung la so phat trien cna to bao them lei so v6i i6 dei chimg (nuoi mai truing 418 IMDM 10% FBS) Trong do, a to be sung djch chiet vi 5% so phat trier chant.' va gin nhu it thy cac tic deng pita djch chiet Vio 15 new, hau nhu town be to bio 218 bj bong va chef town b6 sau 15 ngay; vio c6 xuat hien cac to bio co hinh dang giOng bac) than kinh; nhien se luting rat it, ten met binh nu& cay 25 cm2 chi c6 - to bio 18 15% dich chiet, to bao ding chet nhieu, Tap chi Cong nghe Sinh hQc 6(4): 415-422, 2008 vao Ming xuet hien nhieu bio co hlnh clang giong, to bao than kinh veri so lucmg it va den 15, hau het to bao chat (5 lo 10% dich chiet, to bao chet ttrcmg doi theo th6i gian, den ngay, da co su hien nhieu to bao veri hinh dang rat giong van cac to bao than kinh nhu neuron (Hinh 3A,B,C); so hrqng cac to bao tiep tuc tang len cho den 15 Tiep,tuc nu6i cay cac to bio tren den 21, hau het to bao chet Cac to bao hUCB , MSC thuang duqc phat then mei tnremg „c6 bo sung 10 - 20% FBS, mot so nghien cim be sung EGF va bFGF Khi biet h6a, nhArn tao stress de cam img bier h6a, mei trtrang nuoi cay sir dung ming deo huyet thap (2%); va do, mei truang to bao chet den thieu nhieu phan kich thich tang twang quan trong FBS Nen be sung dich chiet a 1, 5, 10 va 15% deu they co su giant tang twang cua hUCB MSC so ,v6i chimg ruing dO 5%, dich chiet, bao chet it nhet va cho hieu qua thay doi hinh dang re, ref, a', le day Ong di, phi, hop nhat von Itrong chat dich chiet be sung vao lucmg chat mat di giam Ming huyet Phan tram didh chiet qua cao (15%) se lam thay doi ap swat them that, dm mei tin:mg, nen lam to bao chet nhieu Trong mot so lo thi nghiem non,g TO 10% dich chiet c6 xuat hien cac cim to bao giong,nhu,cac neurosphere hinh kin nu6i cay to bao gee than kinh Khi chay RT-PCR danh gia su bieu hien gen nestin (dac cho to bao than kinh) cho thay cac to bio thu nhan tir mAu biet hoa tied dich chi& 10% duang tinh von marker sau 10 cam irng biet hoa (Hinh 3D) Bit hoa hUCB MSC thinh to bio co tim Khi be sung mei truerng cam ling bier hem veri cac Ming dO dich chi& khac deu gay cac phan img cita to bao din yeti mei tnrang mai Cac phan Ung duqc ghi nhan la su bong Uric to bao, su chet, su co cum va hlnh cac cim to bao; ngoiti ming c6 str hinh cac to bao co hinh dang giong to bio ca Su bong tree bao tang den tir 0,5, 1, 2,5, va 10% dich chiet bbee sung va a nOng 40 2,5, va 10% dich chiet, heu nhu toan be to bao chet,vao sau cam img Ming 0,5 va % to bito,phat trier, cham so van den chUng, so lucmg to bio chet it va bit deu hinh cac cam to bao vao 14 - 15 (Hinh 4A), va hen het to bao chet vao 40 Tir 21, a lo biet hoa bang 1% dich chiet, co su xuAt hien met so to bao co hinh dang giting bio co (Hinh 4B) Trong suet 40 theo doi, tit ca cac nieu cac 18 deu khong xuat hien cac to bao co dap Su xuet hien cac to bao co bap IA mot bang chirng manh ma nhieu tac gia a tien hinh ming de bier hoa to bao MSC to bio Ca tim bang 5-azacystidin (XiaoHong Li et al., 2007; Xu et al., 2008) Mau bac, biet h6a sau 21 a to be sung 0,5 va 1% dich chiet duqc ti'en hanh RT-PCR veri gen marker cTnT wing cho to IA° ca tim) cho they chi a 10 veri 1% dich chiet, to bao co bieu hien gen (Hinh 4C) Vi thinh phan dich chi& mei chua kiem sok het, ben canh phan chinh la protein thi dich chiet can nhieu phan khic ma quan la DNA, RNA Cling c6 the Fie thuyet rang su cam img bier hoa la phan, DNA, RNA trong, dich chiet bien nap vao to bao g8c; va do, su bieu hien gen hay hinh dang dat duqc la su bieu hien cim nhung DNA va RNA ,Va neu nhu hien tuqng xity thi su thay doi kieu hinh tren co the la tam than va DNA va RNA ngoai lai chi ten tai to bao mot than gian ngin Nghien dm chua giai quyet duqc van de KET LU IAN Cac to bao gee la nhimg to bio c6 kha nang chuyen doi, biet hoa cac to bao co chirc nang theo dich chiet cim ,m8 dich Trong dich chiet cue mei, nhieu thinh phan protein ngoai bac, va lion bio ton tai Nhung phan cling co the lit you to gay cam img biet Mia to bio gtic theo lcieu hinh cim m0 ay CS ruing deo 400 ttg/m1 protein dich chi& thu tir fay chuOt chua twang thanh, to bac, g8c trung m8 thu tir man cueing fon ngtrai co the, biet hoa cac cam My deo veri hieu qua cao nhat cac ming dO khao sat sau 10 cam img Cac cum qty deo bieu hien cac gen chuyen bier cho to bao tiet insulin nhu Glut-2, Ngn3, Pax4 va Insulin truong bo sung 10% dich chiet nao thai chuOt, su biet hoa cim to bao goc trung m8 cac neurosphere xay manh sau 10 cam ling Cac to bio tir neurosphere bieu hien ducmg tinh vai nestin trtrerng bo sung 1% dich chiet tim heo con, to bao gOc trung mei thay del hinh dang giong hinh Bang to bio co tim sau 21, cam img va bieu hien gen cTnT d4c cho to bao ca tim 419 Pham Van Phac et al Hinh (A) Te bao g6c trung m6 sau thu nhan va khong cam 0,ng biM hea (X20); (B) Te bao g6c trung me sau biM h6a 10 mei trifeng biet h6a chira 400 pg/ml protein dich chiM &vac nhuem yeti dithizone (X10) Cac to bao hUCB MSC sau thu nhan co hinh dung giOng vai nguyen bao sqi Sau biet hoa, cac to bao co va hinh cum to bao gieng yen toy deo Cac cum bat mau de nhuem vbi thuec nhuem dithizone O Actin DOi chieng Nestin Hinh hUCB MSC duvc biet h6a to bao giOng to bao than kinh bang dich chiet (A) Cum to bao grang neurosphere vao 5; (B) Cac to bao CO scyi dal Ong neuron xuat hien vao 7; (C) Cac to bao co nhieu nhanh vai nhanh dai giOng axon xuet hien vao 10 (Ket qua mau be sung dich chiM 10%) (Hinh chup 6' vat kinh X20) (D) KM qua chay RT-PCR vai gen marker Nestin cho to bao than kinh Ket qua cho they to bao bieu hien gen nestin, to bao kh6ng cam irng biM h6a (dei chirrig) kh6ng bleu hien N6ng d6 1% by M Actin MT cTnT DC N6ng d6 0,5% Kh6ng cam Ong MT Actin cTnT DC Actin cTnT DC 600 300 Hinh Ket qua biM haa hUCB MSC to bao ca tim bang dich chiet (A) Com to bao hinh to thi nghiem be sung 1% dich chiet (de phong dal X10); (B) Te bao co hinh clang giOng to bao co, tim &roc ghi nhan vao 21 sau bit h6a 0, to thi nghiem be sung 1% dich chiet (de phOng clai 40X); (C) KM qua RTPCR cho maTh to bao 0, hai le be sung 0,5 va 1% dich chiet cho they chi re 1% CO sty bieu hien dm cTnT 420 Tap chi Cong nghe Sinh hpc 6(4): 415-422, 2008 Ltd cam Chung toi xin chin cam an Benh vien Phu san Hung Vuvng HO,Chi Minh dd cung cap matt mau cu Ong ' ran nguai de tien hanh nghien cieu TAI LIEU THAM KHAO Choi KS, Shin JS, Lee JJ, Kim YS, Kim SB, Kim CW (2005) In vitro trans-differentiation of rat mesenchymal cells into insulin-producing cells by rat pancreatic extract Biochem Biophys Res Commun 330(4): 1299305 Collas P, Taranger CK (2006) Epigenetic reprogramming of nuclei using cell extracts Stem Cell Rev 2(4): 309-317 Gaustad KG, Boquest AC, Anderson BE, Gerdes AM, Collas P (2004) Differentiation of human adipose tissue stem cells using extracts of rat cardiomyocytes Biochem Biophys Res Commun 314(2): 420-427 Hakelien AM, Gaustad KG, Collas P (2006) Modulation of cell fate using nuclear and cytoplasmic extracts Methods Mol Biol 325: 99-114 Hakelien AM, Gaustad KG, Collas P (2004) Transient alteration of cell fate using a nuclear and cytoplasmic extract of an insulinoma cell line Biochem Biophys Res Commun 316(3): 834-841 Liu J, Jin T, Ritchie HH, Smith Al, Clarkson BH (2005) In vitro differentiation and mineralization of human dental pulp cells induced by dentin extract In Vitro Cell Dev Biol Anim 41(7): 232-238 Phan Kim Ngoc, Pham Van Phuc, Tian Bao Ha (2007) Thu nhan va bit h6a to bac) g8c trung mo mau cluing ron ngtr6i Tap chi Phcit trien Khoa hQc & Cong nghe 10(12): 5-10 Phan Kim Ngoac, Pham Van Phfic, Dang Hoang Lam, Tnrang Hai Nhung (2008) Thu nhan to bao g8c da rang tir mau cu8ng ron nguoi Tap chi Y dupe hpc Quoin sir 33(2): 119-124 Qin M, Tai G, Collas P, Polak JM, Bishop AE (2005) Cell extract-derived differentiation of embryonic stem cells Stem Cells 23(6): 712-718 Schimrosczyk K, Song YH, Vykoukal J, Vykoukal D, Bai X, Krohn A, Freyberg S, Alt EU (2008) Liposomemediated transfection with extract from neonatal rat cardiomyocytes induces transdifferentiation of human adipose-derived stem cells into cardiomyocytes Scand J Clin Lab Invest 10: 1-9 Woo C, Li H, Baatnip A, Krause A, Kassem M, Bunger C, Lind M (2006) Effects of bone protein extract on human mesenchymal stem cells proliferation and differentiation J Biomed Mater Res A 79(3): 552-556 Hakelien AM, Landsverk HB, Robl JM, Skalhegg BS, Collas P (2002) Reprogramming fibroblasts to express T-cell functions using cell extracts Nat Biotechnol 20(5): 460-466 XiaoHong Li, XiYong Yu, QiuXiong Lin, ChunYu Deng, ZhiXin Shan, Min Yang, ShuGuang Lin (2007) Bone marrow mesenchymal stem cells differentiate into functional cardiac phenotypes by cardiac microenvironment J Mol Cell Cardiol 42: 295-303 Lee J, Han DJ, Kim SC (2008) In vitro differentiation of human adipose tissue-derived stem cells into cells with pancreatic phenotype by regenerating pancreas extract Biochem Biophys Res Commun 24: 547-551 Xu RX, Chen X, Chen J}1, Han Y, Han BM (2008) Mesenchymal stem cells promote cardiomyocyte hypertrophy in vitro through hypoxia-induced paracrine mechanisms Clin Exp Pharmacol Physiol 10: 176-180 RESEARCH ON DIFFERENTIATION OF HUMAN UMBILICAL CORD BLOOD DERIVED MESENCHYMAL STEM CELL INTO NEURAL-LIKE CELL, CARDIAC MYOCYTE, INSULIN PRODUCING CELL BY TISSUE EXTRACT Pham Van Phuc, Nguyen Dang Khoa, Tran Bao Kiem, Phan Kim Ngoc University of Science, Vietnam National University, HCM City SUMMARY Stem cells can differentiate into functional cells by a lot of agents, such as chemical, physical stimulating, matrix contact, gene transferring, co-culture with functional cell and tissue extract Among them, tissue extract can be commonly used to differentiate stem cells Differentiating stem cells by tissue Author for correspondence: Tel: 84-8-38397719; Fax: 84-8-38967365; E-mail: pknzoc@hcmuns.edu.vn 421 Pham Van Phtic et aL extract not only proves differentiating capacity of that extract but also demonstrate the fate of these stem cells which live in that tissue microenvironment In this research, human umbilical cord blood derived mesenchymal stem cells were differentiated into neural like cells, cardiac myocytes and insulin producing cells using extract from murine fetal brain, porcine fetal heart tissue and immature mouse pancreas Mesenchymal stem cells were derived from human cord blood using previous published procedure Differentiated cell's phenotype was determined by specific morphology, histochemistry staining and the expression of specific genes by RT-PCR The results showed that tissue extract can be used to alter the fate of stem cells to functional cells by using medium with suitable ratio of tissue extract At 400 tg/ml total protein of pancreas extract, mesenchymal stem cells could differentiate into islet with the most efficiency Islet cell expressed some specific genes such as Glut-2, Ngn3, Pax4 and Insulin In medium with 10% brain extract, stem cells differentiated into neurospheres that expressed nestin gene In medium with 1% myocardial extract, stem cells changed the morphology to myocardial cell's morphology and expressed cTnT gene Keywords: Cardiac myocytes, differentiation, human umbilical cord blood, neural like cells, stem cells, tissue extract 422 ... chiet tim heo con, to bao gOc trung mei thay del hinh dang giong hinh Bang to bio co tim sau 21, cam img va bieu hien gen cTnT d4c cho to bao ca tim 419 Pham Van Phac et al Hinh (A) Te bao g6c trung. .. biet hoa cUa bao gac ,trung mo mau cuong rein ngtrai thinh to bao gi8ng to bao than kinh bang dich chiet nao thai chuet, to bio ca tim bang dich chiet tim heo vi to bao fiat insulin bang dich chiet... tuang img cho vies cam img biet h6a to bao tiet insulin, to bao co tim va bao than kinh la moi truing nuoi be sung dich chiet tuyen toy, mei ca tim va mei nao Men twang biet hem duqc thay vii