:: En > En > Networking Grants > Grants > National Grants

Molecular mechanisms of human umbilical cord-derived stem cells integration and vasculogenesis within the cardiac tissue; implications for cardiovascular regeneration (“RE-CORD”).

Young Independent Research Team Grant. Project director: Marilena Lupu; Funding source: Romanian Ministry of Education and Research Framework Program PNCDI-II
Objectives: to investigate the molecular mechanisms of integration into the cardiac tissue of human umbilical cord-derived MSCs and EPCs, as well as their contribution to cardiovascular regeneration by employing in vitro cardiac transplantation models.
During previous and current work, we have established standard operation protocols for characterization of EPCs and MSCs (Fig. 2 and Fig. 3, respectively). For the investigation of cellular and molecular interactions with the cardiac extracellular matrix/myocardium, during integration and vasculogenesis, the cells are cultured, under optimized conditions, with either vital (Fig. 4) or ischemic murine ventricular slices.

The interactions are investigated at gene and protein levels for the assessment of molecules and signalling pathways with determinant roles in stem/progenitor cells chemotaxis, integration, proliferation, and vasculogenesis.

ROMANA ENGLEZA

REZUMAT ABSTRACT

CONCEPT CONCEPT

OBIECTIVE OBJECTIVES

REZULTATE RESULTS

DISEMINARE DISSEMINATION

BIBLIOGRAFIE REFERENCES

Prezentarea proiectului PNCDI-II Nr. 106/2010-2013

Titlul proiectului: “Investigarea mecanismelor moleculare ale integrarii si vasculogenezei aferente celulelor stem umane derivate din cordonul ombilical: implicatii pentru regenerarea cardiovasculara” (acronim, “RE-CORD”)

REZUMAT

INTRODUCERE:

Celulele stem derivate de la nivelul sangelui si matricei cordonului ombilical au fost propuse recent pentru terapia celulara specifica de tesut. De asemenea, sectiunile ventriculare cardiace au fost utilizate cu succes in studii in vitro de transplant de celule stem embrionare. Pana in prezent, aceste sectiuni nu au fost insa folosite in studiul integrarii celulelor stem fetale si a proprietatilor regenerative induse de acestea.

OBIECTIVE:

Investigarea mecanismelor de integrare in tesutul cardiac a celulelor stem mezenchimale (MSC) si a celulelor progenitoare endoteliale (EPC) umane derivate din cordonul ombilical si a contributiei lor la regenerarea cardiovasculara pe modele in vitro de transplant cardiac.

MATERIALE SI METODE:

MSC si EPC, pentru a caror izolare si caracterizare grupul nostru a stabilit deja protocoale standardizate de lucru, au fost co-cultivate, utilizand conditii optimizate, atat cu sectiuni ventriculare cardiace murine viabile, cat si ischemice. Interactiunile dintre MSC/EPC si matricea cardiaca/miocardul murin au fost investigate la nivel genic si proteic pentru stabilirea moleculelor si cailor de semnalizare cu roluri cheie in procesele de migrare (ex. TIE-2, CXCR4, VEGF, VEGFR2), integrare [ex. metaloproteinaze matriceale (MMP), inductorul de MMP (EMMPRIN)], proliferare si vasculogeneza (ex. CD31, CD105, CD144, factorul von Willebrand).

RELEVANTA:

Studiul de fata ofera premize pentru descifrarea mecanismelor de migrare, integrare, proliferare, diferentiere si vasculogeneza, la nivelul tesutului cardiac, induse de celuelele stem derivate din cordonul ombilical, contribuind la dezvoltarea terapiilor regenerative fezabile pentru afectiunile cardiovasculare congenitale si dobandite.

CONCEPT STIINTIFIC

Studiile de cercetare asupra celulele stem au adus o contributie deosebita atat cercetarii fundamentale in domeniul biologiei celulare, cat si clinice, unde aceste studii au fost transferate[1]. Impactul socio-economic tot mai ridicat al bolilor cardiovasculare a directionat eforturile comunitatii stiintifice internationale catre investigarea unor terapii mai eficiente, cum ar fi terapiile celulare specifice de tesut. In timp ce tratamentele actuale ale infarctului miocardic si ale insuficientei cardiace pediatrice sunt menite a induce revascularizarea timpurie si inhibarea pierderii tesutului cardiac nobil[2], terapia celulara se adreseaza restaurarii functiei cardiace pe baza regenearii tesutului cardiovascular. Multiple surse celulare au fost propuse in ultimii ani pentru terapia celulara, insa alegerea celei mai sigure si eficiente surse ramane o problema critica. Pe de o parte, a fost demonstrat deja faptul ca terapia celulara poate contribui la regenerarea vasculara[3-8]. Pe de alta parte, in pofida inducerii unei imbunatatiri modeste a functiei cardiace[9,10], capacitatea de diferentiere a celulelor stem adulte si fetale in cardiomiocite este controversata[4,8,11,12].

Sangele de cordon ombilical (UCB) este utilizat cu succes de aproape doua decenii in tratamentul unor variate afectiuni hematologice, iar bancile ce stocheaza unitati de UCB pot oferi in momentul de fata grefe HLA-compatibile pentru majoritatea pacientilor. Cateva grupuri de cercetatori au propus utilizarea celulelor stem derivate din UCB pentru generarea in vitro a celulelor progenitoare endoteliale (EPC)[13-22]. In concordanta cu aceste studii, cercetari efectuate pe modele animale de transplant de celule hematopoietice sau de ischemie vasculara au aratat faptul ca celulele stem/progenitoare izolate din UCB pot participa la fenomenul de neovascularizatie[23-27], necesara pentru ameliorarea functiei miocardului ischemic[25,26,28,29]. In orice caz, teoria conform careia celulele izolate din UCB pot fi utilizate pentru terapia celulara de inlocuire a tesutului cardiac, in urma diferentierii in cardiomiocite[19,30-33], este foarte controversata, din moment ce aceste celule au esuat in a genera tesut contractil in vivo[34-36]. De aceea, dezvoltarea terapiilor cu celule izolate din UCB, bazate mai degraba pe inducerea efectelor de neovascularizatie, decat pe inducerea diferentierii cardiomiocitare, pentru ameliorarea functiei cardiace, merita o atentie crescuta.

In timp ce celulele stem mezenchimale (MSC) derivate din maduva osoasa hematogena au adus un beneficu terapeutic modest in regenerarea specifica de tesut/organ, a fost postulat recent faptul ca tesutul mezenchimal al cordonului ombilical uman, numit gelatina Wharton (WJ), contine celule stem inzestrate cu proprietati superioare in ceea ce priveste plasticitatea si capacitatea de proliferare[37]. Astfel, celulele derivate din WJ au putut fi diferentiate in celule osoase[37-39], condrocitare[37,40], adipoase[37,39], musculare scheletice[37,41] si neurale[37,42-45]. De aceea, aceste celule au fost recomandate in regenerarea corzilor vocale[46], tratamentul fibrozei hepatice[47], regenerarea musculara[41], inginerizarea cartilajului hialin[40], a tesutului osos[38,39], precum si a valvelor cardiace[48]. In ciuda acestor aplicatii clinice variate, populatiile de celule stem/progenitoare derivate din WJ nu au fost caracterizate complet, iar capacitatea lor de a se integra in tesutul cardiac si de a participa la procesul de neovascularizatie si de imbunatatire a functiei cardiovasculare a fost investigata la un nivel foarte scazut[11,39,49].

A fost evidentiat faptul ca atat membrana bazala, cat si matricea extracelulara trebuie degradate pentru a permite migrarea si integrarea celulelor endoteliale, ca si expansiunea tubilor vasculari; de aceea, trebuie sa existe o coordonare perfecta intre aceste procese pentru initierea si producerea fenomenelor de vasculogeneza si angiogeneza[50,51]. De exemplu, inhibarea degradarii matricei extracelulare limiteaza migrarea celulelor endoteliale si formarea tubilor vasculari[52]. Proteoliza membranei bazale este realizata cu ajutorul catorva familii de enzime, cum ar fi metaloproteinazele matriceale (MMP)[53]. In plus, MMP-urile contribuie la evenimente proteolitice importante in initierea si derularea procesului de angiogeneza[53]. Interesant, a fost aratat faptul ca fortele mecanice induse de catre contractilitatea musculaturii scheletice au fost responsabile de initierea angiogenezei[54] si ca inhibarea activitatii MMP-urilor a anihilat fenomenul de angiogeneza indus in mod specific de stimularea contractilitatii musculare[52]. De asemenea, cai de semnalizare implicand MMP-2 si VEGF, s-au dovedit a fi importante in reglarea fenomenului de angiogeneza[55]. Nu in ultimul rand, grupul nostru a publicat deja date referitoare la prezenta de receptori chemokinci la nivelul celulelor MSC si a EPC derivate din WJ (WJ-MSC and -EPC), cum ar fi VEGFR2, Tie-2 si CXCR4[56,57]. De aceea, rolul acestor receptori in migrarea celulelor stem derivate din WJ, precum si in integrarea lor in matricea cardiaca merita a fi investigat riguros. Mai mult decat atat, tintirea in scop terapeutic a inductorului de MMP cunoscut sub numele de EMMPRIN (Basigin, CD147), o proteina transmembranara inalt glicozilata de suprafata[58], in conditiuni patologice care necesita fie inhibarea, fie promovarea angiogenezei pare a fi o strategie terapeutica promitatoare, insa necesita o intelegere aprofundata a modului sau de actiune si reglare.

In lumina descoperirilor de mai sus, putem conclude ca ne aflam doar la inceputul procesului de a intelege cum matricea extracelulara si micromediul tisular influenteaza procesele de integrare celulara si vasculogeneza/angiogeneza. Prin urmare, este esential sa investigam daca molecule de genul MMP, EMMPRIN si citokine pro-angiogenice joaca roluri esentiale si in interactiunile celulelor stem derivate din UC cu nisa cardiaca, interactiuni ce pot promova migrarea, proliferarea si integrarea lor in tesutul cardiac, ca si fenomenul de vasculogeneza. Cercetari aditionale sunt necesare pentru a elucida daca celulele stem derivate din UC se grefeaza pe termen lung in tesutul cardiac si daca prezinta potential de autoreinnoire cardio-vasculara[37], pentru a stabili valoarea reala a acestor celule pentru domeniul regenerarii cardiovasculare.

Pentru a afla raspunsul la aceste intrebari critice, celulele stem derivate din UC trebuie sa fie riguros examinate pe noi modele de transplant in vitro, ce pot oferi conditii standardizate, controlabile si reproductibile[20,59]. Cunostintele actuale asupra mecanismelor de integrare a celulelor stem adulte in tesutul cardiac si a reconstructiei fiziologice post-transplant sunt inca incomplete. Investigarea acestor fenomene la nivelul culturilor celulare reprezentate de monostraturi de cardiomiocite disociate au generat date valoroase, dar de relevanta limitata pentru a prezice rezultatele transplantului in vivo in structuri celulare multistratificate[60]. Modelele de transplant in vitro ce constau in sectiuni ventriculare viabile[61-63] obtinute din inima de soarece prezinta structuri morfologice conservate, precum si caracteristici electrofiziologice stabile, comparabile situatiei in vivo[61]. Mai mult decat atat, sectiuni ventriculare ischemice, afectate ireversibil prin suprimarea aportului de oxigen si glucoza au fost utilizate ca si model de transplant in vitro ce simuleaza injuriul ischemic[64]. In ciuda valorii deja dovedite a acestor modele experimentale pentru studiile de transplant in vitro de celule stem embrionare, aceste modele nu au fost inca utilizate pentru studii de integrare, chemotaxie, proliferare si diferentiere in nisa cardiaca a celulelor stem fetale si adulte. De aceea, ne-am propus sa utilizam aceste modele pentru a studia mecanismele de integrare a celulelor MSC si a EPC derivate din UC (UC-MSC si -EPC) in tesutul cardiac, ca si mecanismele vasculogenezei induse de acestea.

Studiile de fata pot contribui la standardizarea terapiilor celulare de inlocuire a tesutului cardiovascular pentru diferite maladii congenitale sau dobandite ce afecteaza acest tesut. In cadrul comunitatii medicale, studii ca cel de fata cresc sperantele pentru imbunatatirea statusului general de sanatate al populatiei. Cercetarile de fata pot avea un impact crescut asupra accelerarii transferului direct al rezultatelor experimentale in clinica, pe principiul „from bench to bedside”. Desi nu au fost initiate inca trialuri clinice de regenerare cardiovasculara cu ajutorul celulelor derivate din UC, celulele stem/progenitoare de la nivelul UC sunt considerate o sursa mai sigura pentru terapia celulara alogenica decat cele derivate de la nivelul maduvei osoase hematogene, datorita imunogenicitatii scazute si a proprietatilor imunomodulatorii[65,66]. De asemenea, administrarea sistemica si repetata de celule progenitoare autologe pentru managementul terapeutic al bolilor cardiovasculare s-ar putea dovedi o procedura eficeinta si lipsita de risc. Atat UCB, cat si WJ pot fi stocate fara dificultate la nastere in banci de celule stem/tesuturi, iar diferite populatiile celulare de interes pot fi izolate ulterior in conditii „xenobiotic-free”, pentru administrare in terapia celulara.

OBIECTIV PRINCIPAL

In studiul de fata, ne-am propus sa investigam mecanismele de integrare in tesutul cardiac a UC-MSC si -EPC, precum si a mecanismelor de regenerare vasculara indusa de aceste celule pe modele in vitro de transplant cardiac (Fig. 1).

Fig. 1. Reprezentarea schematica a design-ului experimental constand in cocultivarea UC-MSC si -EPC cu sectiuni ventriculare murine viabile sau ischemice. EPCs, celule progenitoare endoteliale; MSCs, celule stem mezenchimale; MNCs, celule mononucleare; OGD, suprimarea aportului de oxigen si glucoza; UC, cordon ombilical; UCB, sange de cordon ombilical; WJ, gelatina Wharton.

Obiective 2010:

1.) Stabilirea conditiilor de obtinere a sectiunilor ventriculare viabile si ischemice din inima de soarece, adecvate studiilor de transplant in vitro de UC-MSC si -EPC umane ;

2.) Stabilirea conditiilor optime de cocultura a UC-MSC si -EPC umane cu sectiuni ventriculare viabile si ischemice din inima de soarece.

Obiective 2011:

1.) Stabilirea markerilor cu roluri cheie in migrarea UC-MSC si -EPC in cadrul tesutului cardiac viabil/ischemic;

2.) Stabilirea markerilor cu roluri cheie in proliferarea si integrarea UC-MSC si -EPC in cadrul tesutului cardiac viabil/ischemic;

3.) Stabilirea markerilor cu roluri cheie in vasculogeneza indusa de UC-MSC si -EPC la nivelul tesutului cardiac viabil/ischemic.

Obiective 2012:

1.) Studiul si stabilirea cailor de semnalizare implicate in migrarea UC-MSC si -EPC;

2.) Studiul si stabilirea cailor de semnalizare implicate in integrarea UC-MSC si -EPC.

Obiectiv 2013:

Studiul si stabilirea cailor de semnalizare implicate in vasculogeneza indusa de UC-MSC si -EPC.

REZULTATE:

Au fost stabilite conditiile optime de obtinere a sectiunilor ventriculare viabile si ischemice din inima de soarece, adecvate studiilor de transplant in vitro de UC-MSC si -EPC umane. De asemenea, au fost stabilite conditiile optime de cocultura a UC-MSC si -EPC umane cu sectiuni ventriculare viabile si ischemice din inima de soarece[57] (Fig. 2).

Fig. 2. Analiza imunohistochimica a coculturilor (in ziua a 10-a de cocultura) de sectiuni ventriculare embrionare murine viabile (a) cu UC-MSC (b). A, B, C, D, E, F, G. Diferite nivele de integrare a UC-MSC la nivelul sectiunilor ventriculare; H. Control negativ - doar sectiune ventriculara murina; I. Control negativ - doar anticorp secundar. Anticorp policlonal crescut in iepure, anti-pan-cadherina umana - Alexa Fluor 488 (verde); anticorp monoclonal IgG1 crescut in soarece, anti-nuclei umani- Alexa Fluor 555 (rosu); Nuclei - Hoechst (albastru); J. Evaluarea dinamicii integrarii UC-MSC in sectiunile ventriculare viabile pe durata primei saptamani de cocultura, prin masurarea ariilor cu UC-MSC colorate imunohistochimic, la nivelul criosectiunilor seriale.

De asemenea au fost stabiliti markeri cu roluri cheie in migrarea si vasculogeneza UC-MSC si -EPC in cadrul tesutului cardiac viabil/ischemic (Fig. 3)

Fig. 3. Imagini reprezentative ale gelurilor de electroforeza rezultate in urma analizelor de PCR de revers transcriere (A) si Western blotting (B) pentru evaluarea expresiei de mRNA si proteina a diferitelor molecule implicate in chemotaxia si vasculogeneza UC-MSC, comparativ cu o linie comercial disponibila de celule endoteliale umane din vena ombilicala (HUVEC); (C) Analiza de chemotaxie pentru evaluarea activitatii chemotactice a UC-MSC in respuns la actiunea diferitelor chemokine. Rezultatele sunt reprezentate ca medie ± SEM; n = 3; * p

A fost identificat faptul ca UCB-EPC au generat structuri vasculare „tube-like” doar in contact direct cu sectiunile ventriculare viabile (Fig. 4), insa nu si in contact cu sectiunile ventriculare ischemice sau in sistem de cultura „transwell”, atunci cand nu a existat un contact direct intre EPC si sectiuni. De aceea, contactul direct cu tesutul cardiac viabil, mai degraba decat cytokinele solubile (analizate cu ajutorul kitului „Multiplex cytokine assay”, R&D Systems), reprezinta un factor determinant in formarea structurilor vasculare „tube-like” de catre EPC, observatie care deschide noi cai de explorarea a biologiei acestor celule progenitoare.

Fig. 4. Formarea structurilor „tube-like” de catre UCB-EPC in contact direct cu sectiunile ventriculare murine viabile.

Activitatea chemotactica a UCB-EPC a fost evaluata utilizand un analizator celular in timp real (Real-time xCELLigence Cell Analyzer, Roche, Basel, Switzerland) in prezenta supernatantelor derivate de la nivelul sectiunilor ventriculare murine viabile sau ischemice, avand ca si control o linie comercial disponibila de UCB-EPC (endothelial colony-forming cells, ECFC, Lonza Group Ltd., Switzerland). Rezultatele au indicat faptul ca atat EPC cat si ECFC au prezentat o activitate chemotactica crescuta catre supernatantele derivate de la nivelul sectiunilor ventriculare murine ischemice (Fig. 5).

De aceea, elucidarea interactiunilor celulare si moleculare care guverneaza integrarea UCB-EPC si neovascularizatia indusa de acestea este esentiala inaintea aplicarii acestor celule in clinica pentru regenerarea cardiovasculara.

Fig. 5. Evaluarea activitatii chemotactice a UCB-EPC (A) si ECFC (B) in prezenta supernatantelor derivate de la nivelul sectiunilor ventriculare murine viabile sau ischemice.

In plus, a fost demonstrat faptul ca blocarea expresiei de MMP2 la nivel genic si proteic, in UC-MSC, a afectat proprietatile adezive ale acestor celule, precum si integrarea acestora la nivelul sectiunilor ventriculare, sugerand faptul ca MMP2 ar putea juca un rol esential in procesul de remodelare cardiaca indus de UC-MSC (manuscris in preparare).

De asemenea, MSC au exprimat o capacitate de proliferare si de migrare mai mare in urma stimularii cu EMMPRIN recombinant uman (rhEMMPRIN) care a indus si cresterea capacitatii chemotactice a UC-MSC catre sectiunile ventriculare ischemice. Mai mult decat atat, rhEMMPRIN a modulat in sens pozitiv expresia proteica de MMP1, MMP2 si MMP3 si a indus cresterea expresiei genice (Fig. 6) si proteice de VEGF in functie de doza si timpul de expunere la rhEMMPRIN, ceea ce sugereaza faptul ca EMMPRIN ar putea fi o molecula cheie in modularea efectelor regenerative cardiovasculare (manuscris in preparare).

Prin urmare, identificarea unor gene si proteine cheie si a unor noi cai de semnalizare, implicate in interactiunea MMP/EMMPRIN cu matricea extracelulara cardiaca, integrarea cardiaca si remodelarea cardiovasculara, reprezinta un pas esential in modularea efectelor regenerative ale UC-MSC la nivelul tesutului cardiac, in scopul optimizarii protocoalelor clinice de regenerare cardiovasculara.

Fig. 6. Efectul diferitelor doze de rhEMMPRIN si timpi de expunere la rhEMMPRIN asupra expresiei genice de VEGF.

DISEMINAREA REZULTATELOR:

Articole stiintifice generate in cadrul proiectului, pe intreaga durata de desfasurare a acestuia, publicate in/trimise spre publicare la/in preparare pentru reviste internationale indexate ISI:

1. “Integration properties of Wharton's Jelly-derived novel mesenchymal stem cells into ventricular slices of murine hearts”

Authors: Lupu M.*, Khalil M., Andrei E., Iordache F., Pfannkuche K., Neef K., Georgescu A., Buzila C., Brockmeier K., Maniu H., Hescheler J.

Journal: Cellular Physiology and Biochemistry, 28(1): 63-76, 2011.

2. “Effects of plant lectin and extracts on adhesion molecules of endothelial progenitors”

Authors: Iordache F., Iordache C., Pop A., Lupu M.*, Andrei E., Buzila C., Maniu H.

Journal: Central European Journal of Biology, 6(3): 330-341, 2011.

3. "Isolation method and xeno-free culture conditions influence multipotent differentiation capacity of human Wharton’s jelly-derived mesenchymal stem cells”

Authors: Corotchi M.C.*, Popa M.A., Remes A.*, Sima L.E., Gussi I., Lupu Plesu M.*

Journal: Stem Cell Research & Therapy, 4:81, doi: 10.1186/scrt232, 2013.

4. „Modulation of Wharton’s jelly-derived mesenchymal stem cells functional properties by EMMPRIN”

Authors: Popa M.A., Corotchi M.C.*, Remes A.*, Haustein M., Adelmann R., Hescheler J., Khalil M., Plesu (Lupu) M.*

Journal: Cellular Physiology and Biochemistry, in preparation, 2013.

Prezentari Orale la manifestari siintifice internationale:

1. “Cardiac integration and neovascularization properties of umbilical cord-derived progenitor cells” – Oral presentation, Invited Speaker, BIT’s 4^th Annual World Congress of Regenerative Medicine and Stem Cell 2011, Session S421: Cardiovascular Regeneration and Neovascularization, Beijing, China, November 11-13, 2011.

Author: Lupu M.*

2. “Cardiac integration properties of human umbilical cord-derived progenitor cells” – Oral presentation, “RAMSES Annual Workshop” within the European FP7 “RAMSES” project No. 245691/2010-2013, Koeln, Germany, May 11-12, 2011.

Author: Lupu M.*

3. “Gene expression profiling during differentiation of umbilical cord-derived progenitor cells” – Oral presentation, “RAMSES Annual Workshop” within the European FP7 “RAMSES” project No. 245691/2010-2013, Koeln, Germany, May 11-12, 2011.

Authors: Remes A.*, Lupu M.*

4. “Isolation and characterization of endothelial progenitor cells” – Oral presentation, “RAMSES Multiplier Workshop”, within the European FP7 “RAMSES” project No. 245691/2010-2013, Cairo, Egypt, May 3, 2011.

Author: Lupu M.*

5. “Cardiac integration properties of umbilical cord matrix-derived progenitor cells” – Oral presentation, Third International Congress of the Romanian Society for Cell Biology”, Workshop “Stem Cell Biology and Embryology”, Arad, Romania, June 8-12 2011.

Author: Lupu M.*

6. „Molecular mechanisms involved in adult stem and progenitor cells-induced vasculogenesis – Implications for cardiovascular regeneration” – Oral presentation, “Stem Cell Biology and Embryology” Workshop organized within „The Forth International Congress of the Romanian Society for Cell Biology”, Satu Mare, Romania and Debrecen, Hungary, 13-17 Iune 2012.

Autors: Lupu M*., Remes A.*, Popa M.A., Corotchi M.C.*, Simionescu M.

7. “Modulation of Wharton’s jelly-derived mesenchymal stem cells integration into the cardiac tissue” – Oral presentation,“RAMSES Annual Workshop” within the European FP7 “RAMSES” project No. 245691/2010-2013, Bucharest, Romania, 13-16 September 2012.

Authors: Lupu M.*, Popa M.A., Corotchi C.M.*, Remes A.*

8. „Characterization of multipotent differentiation capacity of human Wharton’s jelly-derived mesenchymal stem cells isolated and expanded in xenobiotic-free conditions” – Oral presentation, “RAMSES Annual Workshop” organized within the European FP7 “RAMSES” project No. 245691/2010-2013, Bucharest, Romania, 13-16 September 2012.

Authors: CorotchiM.C.*/ Popa M.A., RemesA.*, SimaL.E., LupuM.*

9.„Modulation of Wharton’s jelly-derived mesenchymal stem cells functional properties by EMMPRIN” – Oral presentation, RAMSES Final Conference”, international conference organized within the European FP7 „RAMSES” project No. 245691/2010-2013, http://www.ramses-eg.com/files/RAMSES%20flyer_A4.pdf, Cairo, Egypt, 7-9 May 2013.

Authors: Popa M.A., Corotchi M.C.*, Remes A.*, Haustein M., Adelmann R., Hescheler J., Khalil M., Plesu (Lupu) M.*

Prezentari sub forma de Poster la manifestari siintifice internationale:

1. “Cardiac integration and vasculogenic properties of human umbilical cord blood- and Wharton's jelly-derived progenitor cells” – Poster presentation, The “1st Cambridge Stem Cell Symposium: Pluripotency and Development”, “Welcome Trust Centre for Stem Cell Research”, Abstract Book pg. 80-81, University of Cambridge, UK, 6-7 July 2011.

Authors: Lupu M.*, Khalil M., Andrei E., Iordache F., Hescheler J.

2. “Gene and protein expression profiling during differentiation of human umbilical cord-derived progenitor cells” – Poster presentation, Third International Congress of the Romanian Society for Cell Biology”, Arad, Romania, 8-12 June 2011.

Authors: Remes A.*, Lupu M.*

3.„Endothelial gene and protein expression in human Wharton’s jelly-derived stem/progenitor cells isolated in different conditions” – Poster presentation,“7th Annual Congress of the German Society for Stem Cell Research”, Leipzig, Germany, 29-30 November 2012.

Authors: Popa M.A./Corotchi M.C.*, Remes A.*, Lupu M.*

4. „Direct cell-to-cell interactions are required for vascular tubes formation by human endothelial progenitor cells in a co-culture system” – Poster presentation, „Forth International Congress of the Romanian Society for Cell Biology”, Satu Mare, Romania and Debrecen, Hungary, 13-17 Iune 2012.

Authors: Remes A.*, Lupu M.*

5.„Characterization of multipotent differentiation capacity of human mesenchymal stem cells isolated and expanded in xenobiotic-free conditions”** – Poster presentation, „Forth International Congress of the Romanian Society for Cell Biology”, Satu Mare, Romania and Debrecen, Hungary, 13-17 Iune 2012.

Authors: Corotchi M.C.*, Remes A.*, Popa M.A., Sima L.E., Lupu M.*

6. “Gene and protein profiling of human Wharton’s Jelly-derived stem/progenitor cells upon exposure to endothelial differentiation conditions”** – Poster presentation, „Forth International Congress of the Romanian Society for Cell Biology”, Satu Mare, Romania and Debrecen, Hungary, 13-17 Iune 2012.

Authors: Popa M.A., Remes A.*, Corotchi M.C.*, Lupu M.*

7.„Isolation method and xeno-free culture conditions influence multipotent differentiation capacity of human Wharton’s jelly-derived mesenchymal stem cells” – Poster presentation,

“RAMSES Final Conference”, international conference organized within the European FP7 „RAMSES” project No. 245691/2010-2013, Cairo, Egypt, 7-9 May 2013.

Authors: Corotchi M.C.*, Popa M.A., Remes A.*, Sima L.E., Gussi I., Plesu (Lupu) M.*

Comunicări ştiinţifice prezentate la conferinţe naţionale:

“Cardiac integration and neovascularization properties of umbilical cord-derived progenitor cells” – Oral presentation, The 41st National Immunology Conference, Timisoara, Romania, 22-24 September 2011, Abstract in “Physiology 2011 Supplement”, ISSN 1223-2076, pg. 29-30.

Authors: Lupu M.*, Khalil M., Iordache F., Andrei E., Pfannkuche K., Buzila C., Neef K., Brockmeier K., Georgescu A., Maniu H., Hescheler J.

*, Membrii echipei proiectului;

**, Prezentarile au obtinut premiul „Best Posters for the Best Young Research Team” conferit de catre “Societatea Romana de Biologie Celulara”.

Referinte bibliografice

1. Thomas E.D., Storb R: Development of the Scientific Foundation of Hematopoietic Cell Transplantation Based on Animal and Human Studies. In Hematopoietic Cell Transplantation. Edited by Thomas E.D., Blume K.G., Forman S. Blackwell Publishing; 1999:1-11.

2. Pillekamp F, Khalil M, Emmel M, Brockmeier K, Hescheler J: Stem cells in pediatric heart failure. Minerva Cardioangiol 2008, 56: 335-348.

3. Pesce M, Orlandi A, Iachininoto MG, Straino S, Torella AR, Rizzuti V et al.: Myoendothelial differentiation of human umbilical cord blood-derived stem cells in ischemic limb tissues. Circ Res 2003, 93: e51-e62.

4. Jackson KA, Majka SM, Wang H, Pocius J, Hartley CJ, Majesky MW et al.: Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 2001, 107: 1395-1402.

5. Au P, Tam J, Fukumura D, Jain RK: Bone marrow derived mesenchymal stem cells facilitate engineering of long-lasting functional vasculature. Blood 2008.

6. Au P, Daheron LM, Duda DG, Cohen KS, Tyrrell JA, Lanning RM et al.: Differential in vivo potential of endothelial progenitor cells from human umbilical cord blood and adult peripheral blood to form functional long-lasting vessels. Blood 2008, 111: 1302-1305.

7. Madeddu P, Emanueli C, Pelosi E, Salis MB, Cerio AM, Bonanno G et al.: Transplantation of low dose CD34+KDR+ cells promotes vascular and muscular regeneration in ischemic limbs. FASEB J 2004, 18: 1737-1739.

8. Dengler TJ, Katus HA: Stem cell therapy for the infarcted heart ("cellular cardiomyoplasty"). Herz 2002, 27: 598-610.

9. Henning RJ, bu-Ali H, Balis JU, Morgan MB, Willing AE, Sanberg PR: Human umbilical cord blood mononuclear cells for the treatment of acute myocardial infarction. Cell Transplant 2004, 13: 729-739.

10. Kim BO, Tian H, Prasongsukarn K, Wu J, Angoulvant D, Wnendt S et al.: Cell transplantation improves ventricular function after a myocardial infarction: a preclinical study of human unrestricted somatic stem cells in a porcine model. Circulation 2005, 112: I96-104.

11. Pereira WC, Khushnooma I, Madkaikar M, Ghosh K: Reproducible methodology for the isolation of mesenchymal stem cells from human umbilical cord and its potential for cardiomyocyte generation. J Tissue Eng Regen Med 2008, 2: 394-399.

12. Nygren JM, Jovinge S, Breitbach M, Sawen P, Roll W, Hescheler J et al.: Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation. Nat Med 2004, 10: 494-501.

13. Hristov M, Erl W, Weber PC: Endothelial progenitor cells: mobilization, differentiation, and homing. Arterioscler Thromb Vasc Biol 2003, 23: 1185-1189.

14. Lu X, Baudouin SV, Gillespie JI, Anderson JJ, Dickinson AM: A comparison of CFU-GM, BFU-E and endothelial progenitor cells using ex vivo expansion of selected cord blood CD133(+) and CD34(+) cells. Cytotherapy 2007, 9: 292-300.

15. Wu X, Lensch MW, Wylie-Sears J, Daley GQ, Bischoff J: Hemogenic Endothelial Progenitors Cells Isolated from Human Umbilical Cord Blood. Stem Cells 2007.

16. Schmidt D, Asmis LM, Odermatt B, Kelm J, Breymann C, Gossi M et al.: Engineered living blood vessels: functional endothelia generated from human umbilical cord-derived progenitors. Ann Thorac Surg 2006, 82: 1465-1471.

17. Chiu B, Wan JZ, Abley D, Akabutu J: Induction of vascular endothelial phenotype and cellular proliferation from human cord blood stem cells cultured in simulated microgravity. Acta Astronaut 2005, 56: 918-922.

18. Murohara T: Therapeutic vasculogenesis using human cord blood-derived endothelial progenitors. Trends Cardiovasc Med 2001, 11: 303-307.

19. Bonanno G, Mariotti A, Procoli A, Corallo M, Rutella S, Pessina G et al.: Human cord blood CD133+ cells immunoselected by a clinical-grade apparatus differentiate in vitro into endothelial- and cardiomyocyte-like cells. Transfusion 2007, 47: 280-289.

20. Wu KH, Zhou B, Lu SH, Feng B, Yang SG, Du WT et al.: In vitro and in vivo differentiation of human umbilical cord derived stem cells into endothelial cells. J Cell Biochem 2007, 100: 608-616.

21. Ingram DA, Mead LE, Tanaka H, Meade V, Fenoglio A, Mortell K et al.: Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. Blood 2004, 104: 2752-2760.

22. Yoder MC, Mead LE, Prater D, Krier TR, Mroueh KN, Li F et al.: Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals. Blood 2007, 109: 1801-1809.

23. Droetto S, Viale A, Primo L, Jordaney N, Bruno S, Pagano M et al.: Vasculogenic potential of long term repopulating cord blood progenitors. FASEB J 2004, 18: 1273-1275.

24. Crisa L, Cirulli V, Smith KA, Ellisman MH, Torbett BE, Salomon DR: Human cord blood progenitors sustain thymic T-cell development and a novel form of angiogenesis. Blood 1999, 94: 3928-3940.

25. Botta R, Gao E, Stassi G, Bonci D, Pelosi E, Zwas D et al.: Heart infarct in NOD-SCID mice: therapeutic vasculogenesis by transplantation of human CD34+ cells and low dose CD34+KDR+ cells. FASEB J 2004, 18: 1392-1394.

26. Ott I, Keller U, Knoedler M, Gotze KS, Doss K, Fischer P et al.: Endothelial-like cells expanded from CD34+ blood cells improve left ventricular function after experimental myocardial infarction. FASEB J 2005, 19: 992-994.

27. Yang C, Zhang ZH, Li ZJ, Yang RC, Qian GQ, Han ZC: Enhancement of neovascularization with cord blood CD133+ cell-derived endothelial progenitor cell transplantation. Thromb Haemost 2004, 91: 1202-1212.

28. Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, Wang J et al.: Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 2001, 7: 430-436.

29. Kawamoto A, Gwon HC, Iwaguro H, Yamaguchi JI, Uchida S, Masuda H et al.: Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation 2001, 103: 634-637.

30. Prat-Vidal C, Roura S, Farre J, Galvez C, Llach A, Molina CE et al.: Umbilical cord blood-derived stem cells spontaneously express cardiomyogenic traits. Transplant Proc 2007, 39: 2434-2437.

31. Nishiyama N, Miyoshi S, Hida N, Uyama T, Okamoto K, Ikegami Y et al.: The significant cardiomyogenic potential of human umbilical cord blood-derived mesenchymal stem cells in vitro. Stem Cells 2007, 25: 2017-2024.

32. Vanelli P, Beltrami S, Cesana E, Cicero D, Zaza A, Rossi E et al.: Cardiac precursors in human bone marrow and cord blood: in vitro cell cardiogenesis. Ital Heart J 2004, 5: 384-388.

33. Cheng F, Zou P, Yang H, Yu Z, Zhong Z: Induced differentiation of human cord blood mesenchymal stem/progenitor cells into cardiomyocyte-like cells in vitro. J Huazhong Univ Sci Technolog Med Sci 2003, 23: 154-157.

34. Ma N, Ladilov Y, Kaminski A, Piechaczek C, Choi YH, Li W et al.: Umbilical cord blood cell transplantation for myocardial regeneration. Transplant Proc 2006, 38: 771-773.

35. Rabald S, Marx G, Mix B, Stephani C, Kamprad M, Cross M et al.: Cord blood cell therapy alters LV remodeling and cytokine expression but does not improve heart function after myocardial infarction in rats. Cell Physiol Biochem 2008, 21: 395-408.

36. Moelker AD, Baks T, Wever KM, Spitskovsky D, Wielopolski PA, van Beusekom HM et al.: Intracoronary delivery of umbilical cord blood derived unrestricted somatic stem cells is not suitable to improve LV function after myocardial infarction in swine. J Mol Cell Cardiol 2007, 42: 735-745.

37. Troyer DL, Weiss ML: Wharton's jelly-derived cells are a primitive stromal cell population. Stem Cells 2008, 26: 591-599.

38. Hou T, Xu J, Wu X, Xie Z, Luo F, Zhang Z et al.: Umbilical Cord Wharton's Jelly: A New Potential Cell Source of Mesenchymal Stromal Cells for Bone Tissue Engineering. Tissue Eng Part A 2009.

39. Wang HS, Hung SC, Peng ST, Huang CC, Wei HM, Guo YJ et al.: Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord. Stem Cells 2004, 22: 1330-1337.

40. Wang L, Tran I, Seshareddy K, Weiss ML, Detamore MS: A Comparison of Human Bone Marrow-Derived Mesenchymal Stem Cells and Human Umbilical Cord-Derived Mesenchymal Stromal Cells for Cartilage Tissue Engineering. Tissue Eng Part A 2009.

41. Conconi MT, Burra P, Di Liddo R, Calore C, Turetta M, Bellini S et al.: CD105(+) cells from Wharton's jelly show in vitro and in vivo myogenic differentiative potential. Int J Mol Med 2006, 18: 1089-1096.

42. Zhang L, Zhang HT, Hong SQ, Ma X, Jiang XD, Xu RX: Cografted Wharton's Jelly Cells-derived Neurospheres and BDNF Promote Functional Recovery After Rat Spinal Cord Transection. Neurochem Res 2009.

43. Mitchell KE, Weiss ML, Mitchell BM, Martin P, Davis D, Morales L et al.: Matrix cells from Wharton's jelly form neurons and glia. Stem Cells 2003, 21: 50-60.

44. Yang CC, Shih YH, Ko MH, Hsu SY, Cheng H, Fu YS: Transplantation of human umbilical mesenchymal stem cells from Wharton's jelly after complete transection of the rat spinal cord. PLoS One 2008, 3: e3336.

45. Weiss ML, Medicetty S, Bledsoe AR, Rachakatla RS, Choi M, Merchav S et al.: Human umbilical cord matrix stem cells: preliminary characterization and effect of transplantation in a rodent model of Parkinson's disease. Stem Cells 2006, 24: 781-792.

46. Chan RW, Rodriguez ML, McFetridge PS: The human umbilical vein with Wharton's jelly as an allogeneic, acellular construct for vocal fold restoration. Tissue Eng Part A 2009.

47. Tsai PC, Fu TW, Chen YM, Ko TL, Chen TH, Shih YH et al.: The therapeutic potential of human umbilical mesenchymal stem cells from Wharton's jelly in the treatment of rat liver fibrosis. Liver Transpl 2009, 15: 484-495.

48. Schmidt D, Mol A, Odermatt B, Neuenschwander S, Breymann C, Gossi M et al.: Engineering of biologically active living heart valve leaflets using human umbilical cord-derived progenitor cells. Tissue Eng 2006, 12: 3223-3232.

49. Wu KH, Zhou B, Yu CT, Cui B, Lu SH, Han ZC et al.: Therapeutic potential of human umbilical cord derived stem cells in a rat myocardial infarction model. Ann Thorac Surg 2007, 83: 1491-1498.

50. Egginton S, Zhou AL, Brown MD, Hudlicka O: Unorthodox angiogenesis in skeletal muscle. Cardiovasc Res 2001, 49: 634-646.

51. Hansen-Smith FM, Hudlicka O, Egginton S: In vivo angiogenesis in adult rat skeletal muscle: early changes in capillary network architecture and ultrastructure. Cell Tissue Res 1996, 286: 123-136.

52. Haas TL, Milkiewicz M, Davis SJ, Zhou AL, Egginton S, Brown MD et al.: Matrix metalloproteinase activity is required for activity-induced angiogenesis in rat skeletal muscle. Am J Physiol Heart Circ Physiol 2000, 279: H1540-H1547.

53. Prior BM, Yang HT, Terjung RL: What makes vessels grow with exercise training? J Appl Physiol 2004, 97: 1119-1128.

54. Brown MD, Hudlicka O: Modulation of physiological angiogenesis in skeletal muscle by mechanical forces: involvement of VEGF and metalloproteinases. Angiogenesis 2003, 6: 1-14.

55. Park JH, Han HJ: Caveolin-1 plays important role in EGF-induced migration and proliferation of mouse embryonic stem cells: involvement of PI3K/Akt and ERK. Am J Physiol Cell Physiol 2009, 297: C935-C944.

56. Lupu M, Khalil M, Iordache F, Andrei E, Pfannkuche K, Spitkovsky D et al.: Direct Contact of Umbilical Cord Blood Endothelial Progenitors with Living Cardiac Tissue is a Requirement for Vascular Tube-like Structures Formation. J Cell Mol Med 2010.

57. Lupu M, Khalil M, Andrei E, Iordache F, Pfannkuche K, Neef K et al.: Integration properties of Wharton's jelly-derived novel mesenchymal stem cells into ventricular slices of murine hearts. Cell Physiol Biochem 2011, 28: 63-76.

58. Muramatsu T, Miyauchi T: Basigin (CD147): a multifunctional transmembrane protein involved in reproduction, neural function, inflammation and tumor invasion. Histol Histopathol 2003, 18: 981-987.

59. Can A, Karahuseyinoglu S: Concise Review: Human Umbilical Cord Stroma with Regard to the Source of Fetus-Derived Stem Cells. Stem Cells 2007.

60. Sys SU, De Keulenaer GW, Brutsaert DL: Reappraisal of the multicellular preparation for the in vitro physiopharmacological evaluation of myocardial performance. Adv Exp Med Biol 1998, 453: 441-450.

61. Pillekamp F, Reppel M, Dinkelacker V, Duan Y, Jazmati N, Bloch W et al.: Establishment and characterization of a mouse embryonic heart slice preparation. Cell Physiol Biochem 2005, 16: 127-132.

62. Pillekamp F, Halbach M, Reppel M, Rubenchyk O, Pfannkuche K, Xi JY et al.: Neonatal murine heart slices. A robust model to study ventricular isometric contractions. Cell Physiol Biochem 2007, 20: 837-846.

63. Halbach M, Pillekamp F, Brockmeier K, Hescheler J, Muller-Ehmsen J, Reppel M: Ventricular slices of adult mouse hearts--a new multicellular in vitro model for electrophysiological studies. Cell Physiol Biochem 2006, 18: 1-8.

64. Zhang JG, Ghosh S, Ockleford CD, Galinanes M: Characterization of an in vitro model for the study of the short and prolonged effects of myocardial ischaemia and reperfusion in man. Clin Sci (Lond) 2000, 99: 443-453.

65. Rocha V, Wagner JE, Jr., Sobocinski KA, Klein JP, Zhang MJ, Horowitz MM et al.: Graft-versus-host disease in children who have received a cord-blood or bone marrow transplant from an HLA-identical sibling. Eurocord and International Bone Marrow Transplant Registry Working Committee on Alternative Donor and Stem Cell Sources. N Engl J Med 2000, 342: 1846-1854.

66. Weiss ML, Anderson C, Medicetty S, Seshareddy KB, Weiss RJ, VanderWerff I et al.: Immune properties of human umbilical cord Wharton's jelly-derived cells. Stem Cells 2008, 26: 2865-2874.

PNCDI-II Project No. 106/2010-2013

Project title: “Molecular mechanisms of human umbilical cord-derived stem cells integration and vasculogenesis within the cardiac tissue; implications for cardiovascular regeneration (acronym, “RE-CORD”)

ABSTRACT

INTRODUCTION:

The umbilical cord (UC) blood and matrix-derived stem cells have been recently proposed for tissue-specific cell replacement therapy. Cardiac slices with morphological and functional conserved structures have been successfully used for in vitro cardiac transplantation studies of embryonic stem cells. Thus far, cardiac slices have not been used to study the integration and regenerative properties of fetal stem cells.

OBJECTIVE:

Investigation of the mechanisms of human UC-derived mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) integration into the cardiac tissue and their contribution to cardiovascular regeneration by employing in vitro cardiac transplantation models.

MATERIALS AND METHODS:

MSCs and EPCs, for whose isolation and characterization we have already established standard operation protocols, were cocultured, under optimized conditions, with either vital or ischemic murine ventricular slices. Interaction of MSCs and EPCs with the cardiac extracellular matrix/myocardium were investigated at gene and protein levels for assessment of molecules and signaling pathways with determinant roles in MSCs/EPCs homing (i.e. Tie-2, CXCR4, VEGF, VEGFR2), integration [i.e. matrix metalloproteinases (MM¨Ps), MMPs inducer (EMMPRIN)], proliferation and vasculogenesis [i.e. CD31, CD105, CD144, von Willebrand factor).

RELEVANCE:

The studies described here have premises to depict the mechanisms of UC-derived stem cells migration, integration, proliferation, differentiation, and vasculogenesis into the cardiac tissue, contributing to the development of reliable cell replacement therapies for cardiovascular repair of congenital and acquired diseases.

SCIENTIFIC CONCEPT

Stem cell research studies have represented a valuable contribution to both fundamental biology of stem cells and life-saving clinical therapy applications[1]. The increasing socio-economic impacts of cardiovascular diseases have directed scientific community’s research towards investigation of more efficient therapies for these diseases, such as cell replacement therapies. While current treatment of myocardial infarction and pediatric heart failure focuses on early revascularization and medical treatment for the inhibition of further cardiomyocyte loss[2], the approach of stem cell therapy aims at restoration of cardiac function by regenerating the cardiovascular tissue. Several cell sources have been suggested for cellular therapies and choosing the safest and the most efficient one is a critical issue. It has been already shown that cell therapy employing various adult stem/progenitor cells has the potential to provide functional endothelium[3-8]; however, despite the improvements seen in cardiac function[9,10], their differentiation to cardiomyocytes has been controversial[4,8,11,12].

The umbilical cord (UC) blood (UCB) has been successfully applied for two decades to treat a variety of hematological disorders, and cord blood banks could provide now HLA-matched grafts for the majority of patients. Several groups addressed the use of UCB-derived stem cellsfor in vitro generation of endothelial progenitor cells (EPCs)[13-22]. In line with this, research studies performed on animal models of hematopoietic cell transplantation or vascular ischemia have shown that transplanted UCB-derived stem/progenitor cells could participate to neovascularization[23-27] needed for improvement of ischemic heart function[25,26,28,29]. However, the suggestion that UCB cells can be used for cardiac replacement therapy upon differentiation into cardiomyocytes[19,30-33] has been controversial, as these cells failed to form contractile tissue in vivo[34-36]. Therefore, the development of UCB-derived stem cell therapies relying on neovascularization rather than myocyte differentiation, to improve impaired cardiac function following cardiomyocyte loss, deserves further attention.

Whereas adult bone marrow-derived mesenchymal stem cells (MSCs) have shown limited therapeutic benefit for organ regeneration, it has been postulated that human umbilical cord mesenchymal tissue, namely, Wharton’s jelly (WJ), contains stem cells that might be endowed with superior plasticity properties and a greater expansion capability[37]. WJ-derived cells were able to differentiate into bone[37-39], cartilage[37,40], adipose[37,39], muscle[37,41], and neural[37,42-45] cells. Therefore, these cells have been recommended for cell therapy in vocal folds restoration[46], for treatment of liver fibrosis[47], myogenic regeneration[41], hyaline cartilage engineering[40], bone tissue engineering[38,39], and living heart valve leaflets engineering[48]. However, despite of these wide range of potential clinical applications, WJ-derived stem/progenitor cell populations have not been fully characterized, and their ability to integrate into the cardiac tissue, participate to neovascularization and improve cardiovascular function has been investigated at a very low extent[11,39,49].

It has been reported that the basement membrane and the extracellular matrix must be degraded to permit migration and integration of endothelial cells, as well as expansion of the developing tube, and thus there must be a coordination of multiple processes for vasculogenesis and sprouting angiogenesis [50,51]. For example, inhibiting the degradation of the extracellular matrix limits endothelial cell migration and tube formation[52]. Proteolysis of the basement membrane is achieved by several families of enzymes, such as matrix metalloproteinases (MMPs)[53]. Activation of MMPs appear to be important proteolytic events facilitating angiogenesis[53]. Interestingly, it has been shown that contraction of the skeletal muscle led to mechanical forces acting on the tissue that could initiate angiogenesis[54], and that inhibition of MMP activity eliminated the angiogenesis typically induced by muscle stimulation[52]. Also, signaling pathways involving MMP-2 and VEGF, proved also to be important in regulation of angiogenesis[55]. Nevertheless, we have already reported the presence of chemokine receptors on WJ-derived MSCs and EPCs, such as VEGFR2, Tie-2, and CXCR4[56,57]. Therefore, the role of these receptors in WJ-derived stem cell homing and integration into the cardiac matrix deserves further attention. Furthermore, targeting the MMPs inducer EMMPRIN (Basigin, CD147), a highly glycosylated cell surface transmembrane protein[58], in pathological conditions that require either inhibition or promotion of angiogenesis appears a promising future therapeutic strategy, but requires a better understanding of its mode of action and regulation.

We are only beginning to understand how extracellular matrix and tissue environment influence cell integration and vasculogenesis/angiogenesis. Thus, it is vital to further investigate whether molecules such as MMPs, EMMPRIN and angiogenic cytokines play essential roles in UC-derived stem cells interactions with the cardiac niche that promote their migration, proliferation, integration and induced-vasculogenesis. Further work is needed to gain more insights into the biology of UC-derived stem cells and determine whether they engraft long-term into the cardiac tissue and display self-renewal cardiovascular-specific potency[37], in order to elucidate the real value of UC-derived stem cells for clinical cardiovascular regeneration.

To answer these critical questions, UC-derived stem cells deserve to be thoroughly examined in novel in vitro transplantation models that could provide well controlled and reproducible environments[20,59]. Current knowledge of the mechanisms of adult stem cell integration into the cardiac tissue and physiological reconstitution after transplantation is still incomplete. The investigation of these phenomena at the level of cell cultures consisting of monolayers of dissociated cardiomyocytes have generated valuable data, but of limited relevance to predict the outcomes of in vivo transplantation into multicellular tissue structures[60]. The in vitro transplantation models consisting of vital ventricular tissue slices[61-63] of murine hearts have shown morphologically conserved structures and stable electrophysiological characteristics, comparable to the in vivo counterparts[61]. Furthermore, non-vital, non-contractile ventricular slice preparations damaged irreversibly by oxygen and glucose deprivation have been used as an in vitro transplantation model that simulated severe ischemic injury[64]. Despite the already proven value of these models for in vitro embryonic stem cells transplantation studies, they have not yet been used for studies of fetal and adult stem cells integration, chemotaxis, proliferation, differentiation and survival into the cardiac niche. Therefore, we proposed to take advantage of the above models to start addressing these issues by investigating the mechanisms of UC-MSCs and -EPCs integration into the cardiac tissue and their participation to vascular regeneration.

The present studies have premises to contribute to standardization of reliable cell replacement therapies for cardiovascular repair of congenital and acquired defects. Within the medical community, studies resembling that proposed here raise hopes for the improvement of population general health status. The present studies might have a significant potential for speeding clinical translation of experimental results from bench to bedside. Although clinical trials of cardiovascular regeneration involving the use of UC-derived cells have not been yet undertaken, UC-derived stem/progenitor cells are considered to be a safer source for allogeneic cell therapy than bone marrow-derived stem cells, due to their low immunogenicity and high immunomodulatory properties[65,66]. In addition, systemic and repeated delivery of autologous progenitor cells for the therapeutic management of cardio-vascular diseases would be a simple and safe procedure in human patients. Both UCB and WJ could be easily stored at birth in tissue banking services, and specific clinical-grade cell populations isolated afterwards, as needed, for cell therapy purposes.

MAIN OBJECTIVE

We proposed to investigate the mechanisms of integration of human UC-MSCs and -EPCs into the cardiovascular tissue and their contribution to vascular regeneration by employing in vitro cardiac transplantation models (Fig. 1).

Fig. 1. Schematic representation of the experimental design consisting of coculture of UC-derived MSCs/EPCs with either murine living or ischemic ventricular slices. EPCs, endothelial progenitor cells; MSCs, mesenchymal stem cells; MNCs, mononuclear cells; OGD, oxygen and glucose deprivation; UC, umbilical cord; UCB, umbilical cord blood; WJ, Wharton’s jelly.

Objectives 2010:

1.) Establishment of experimental conditions for generation of viable and ischemic murine ventricular slices, suitable for in vitro transplantation studies involving the use of human UC-MSCs and -EPCs;

2.) Establishment of the optimal conditions for coculture of human UC-MSCs and -EPCs with viable and ischemic murine ventricular slices.

Objectives 2011:

1.) Establishment of markers with key roles in UC-MSCs and -EPCs chemotaxis towards the viable/ischemic cardiac tissue;

2.) Establishment of markers with key roles in UC-MSCs and -EPCs proliferation and integration within the viable/ischemic cardiac tissue;

3.) Establishment of markers with key roles in UC-MSCs and -EPCs vasculogenesis within the viable/ischemic cardiac tissue.

Objectives 2012:

1.) Investigation of molecular mechanisms/signaling pathways involved in chemotaxis of UC-MSCs and -EPCs.

2.) Investigation of molecular mechanisms/signaling pathways involved in integration of UC-MSCs and -EPCs.

Objectiv 2013:

Investigation of molecular mechanisms/signaling pathways involved in vasculogenesis induced by UC-MSCs and -EPCs.

RESULTS:

We established the optimal experimental conditions for generation of viable and ischemic murine ventricular slices, suitable for in vitro transplantation studies involving the use of human UC-MSCs and -EPCs. In addition, we established the optimal conditions for coculture of human UC-MSCs and -EPCs with viable and ischemic murine ventricular slices[57] (Fig. 2).

Fig. 2. Immunohistochemistry analyses of 10-day cocultures of murine embryonic ventricular living slices(a)with UC-MSCs(b). A, B, C, D, E, F, G. Different levels of integration of UC-MSCs into the murine embryonic ventricular living slices; H. Negative control - murine ventricular slice only; I. Negative control - secondary antibodies only. Polyclonal rabbit anti-human pan-cadherin - Alexa Fluor 488 (green); Monoclonal IgG1 mouse anti-human nuclei - Alexa Fluor 555 (red); Nuclei - Hoechst (blue); J. Assessment of the dynamics of UC-MSCs integration into the living ventricular slice preparations during the first week of coculture, by measurement of areas with immunohistochemically stained UC-MSCs at the level of serial cryosections.

In addition, we identified the markers with key roles in migration and vasculogenesis induced by UC-MSC and -EPC at the level of viable/ischemic cardiac tissue (Fig. 3)

Fig. 3. Representative images of reverse transcription polymerase chain reaction (A) and Western blotting (B) analysis for the assessment of mRNA and protein expression of molecules involved in UC-MSCs chemotaxis and vasculogenesis, as compared to a commercially available line of human umbilical vein endothelial cells (HUVECs); (C) Chemotaxis assay for the evaluation of UC-MSCs chemotactic activity in response to various chemokines. Results are represented as mean ± SEM; n = 3; * p

We also identified that UCB-EPCs generated „tube-like” structures only in direct contact with the viable ventricular slices (Fig. 4), but not in direct contact with the ischemic ventricular slices or in the “tranwell” system where there was no direct contact between the UCB-EPCs and the slices. Therefore, the direct contact with the viable cardiac tissue rather than the soluble cytokines (analyzed via „Multiplex cytokine assay”, R&D Systems), represent a determinant factor in vascular “tube-like” structures formation by UCB-EPCs, observation that opens new insights into the biology of these progenitor cells.

Fig. 4. Formation of vascular “tube-like” structurilor „tube-like” structures by UCB-EPCs in direct contact with the viable murine ventricular slices.

The chemotactic activity of UCB-EPCs was evaluated by using a real-time cell analyzer (Real-time xCELLigence Cell Analyzer, Roche, Basel, Switzerland) in the presence of supernatants derived from viable or ischemic murine ventricular slices, having as control a commercially available UCB-EPCs line (endothelial colony-forming cells, ECFCs, Lonza Group Ltd., Switzerland). The results indicated that both UCB-EPCs and ECFCs presented an increased chemotaxis towards the supernatants derived from ischemic ventricular slices (Fig. 5).

Therefore, the elucidation of cellular and molecular interactions that govern the integration of UCB-EPCs and their induced neovascularization at the level of the cardiac tissue is essential before applying these cells in the clinic for cardiovascular regeneration purposes.

Fig. 5. Evaluation of chemotactic activity of UCB-EPCs (A) si ECFC (B) in the presence of supernatants derived from viable or ischemic murine ventricular slices.

Furthermore, we demonstrated that blocking of MMP2 at gene and protein levels in UC-MSCs leads to impaired cell adhesion and integration into ventricular slices, suggesting that MMP2 could play an essential role in cardiac remodeling my UC-MSCs (manuscript in preparation).

In addition, we demonstrated that UC-MSCs exhibited a higher proliferation and migration rate in response to stimulation with the MMPs inducer recombinant human (rh) EMMPRIN that increased UC-MSCs chemotaxis towards ischemic ventricular slices. Moreover, rhEMMPRIN upregulated MMP1/2/3 protein expression, as well as VEGF gene (Fig. 6) and protein expression in a dose-time dependent manner, suggesting its value for modulating cardiovascular regeneration outcomes (manuscript in preparation).

Therefore, identification of key genes and proteins involved in the interaction of MMPs/EMMPRIN with cardiac extracellular matrix, cardiac integration, and cardiovascular remodeling could lead to modulation of UC-MSCs integration, resulting in optimized clinical cardiovascular regeneration.

Fig. 6. The effect of various doses and exposure times of/to rhEMMPRIN on VEGF gene expression.

RESULTS DISSEMINATION:

Scientific articles, generated within the project, published in/submitted to/in preparation for ISI international journals: