Embryonic stem cells, derived either after in vitro fertilization or nuclear transfer, prolong survival of semiallogeneic heart transplants.

Abstract

Tolerance induction toward allogeneic organ grafts represents one of the major aims of transplantation medicine. Stem cells are promising candidates for promoting donor-specific tolerance. In this study, we investigated the immunomodulatory properties of murine embryonic stem cells (ESCs), obtained either by in vitro fertilization (IVF-ESCs) or by nuclear transfer (NT-ESCs), in heart transplant mouse models. IVF-ESCs did not prolong the survival of fully allogeneic cardiac transplants but significantly prolonged the survival of semiallogeneic hearts from the same ESC donor strain for >100 d in 44% of the animals. However, 28% of transplanted animals infused with IVF-ESCs experienced development of a teratoma. NT-ESCs similarly prolonged semiallogeneic heart graft survival (>100 d in 40% of the animals) but were less teratogenic. By in vitro studies, IVF-ESC and NT-ESC immunoregulation was mediated both by cell contact-dependent mechanisms and by the release of soluble factors. By adding specific inhibitors, we identified PGE(2) as a soluble mediator of ESC immunoregulation. Expansion of regulatory T cells was found in lymphoid organs and in the grafts of IVF-ESC- and NT-ESC-tolerized mice. Our study demonstrates that both IVF-ESCs and NT-ESCs modulate recipient immune response toward tolerance to solid organ transplantation, and that NT-ESCs exhibit a lower tendency for teratoma formation. Because NT-ESCs are obtained by NT of a somatic cell from living individuals into an enucleated oocyte, they could represent a source of donor-derived stem cells to induce tolerance to solid organ allograft.

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The Journal of Immunology
Embryonic Stem Cells, Derived Either after In Vitro
Fertilization or Nuclear Transfer, Prolong Survival of
Semiallogeneic Heart Transplants
Barbara Imberti,*,1Federica Casiraghi,†,1Daniela Cugini,†Nadia Azzollini,†
Paola Cassis,†Marta Todeschini,†Samantha Solini,†Vittorio Sebastiano,‡,x,2
Maurizio Zuccotti,{Silvia Garagna,‡,xCarlo Alberto Redi,‖Marina Noris,†
Marina Morigi,* and Giuseppe Remuzzi*,†,#
Tolerance induction toward allogeneic organ grafts represents one of the major aims of transplantation medicine. Stem cells are
promising candidates for promoting donor-specific tolerance. In this study, we investigated the immunomodulatory properties of
murine embryonic stem cells (ESCs), obtained either by invitro fertilization (IVF-ESCs) or by nuclear transfer (NT-ESCs), in heart
transplant mouse models. IVF-ESCs did not prolong the survival of fully allogeneic cardiac transplants but significantly prolonged
the survival of semiallogeneic hearts from the same ESC donor strain for >100 d in 44% of the animals. However, 28% of
transplanted animals infused with IVF-ESCs experienced development of a teratoma. NT-ESCs similarly prolonged semialloge-
neic heart graft survival (>100 d in 40% of the animals) but were less teratogenic. By in vitro studies, IVF-ESC and NT-ESC
immunoregulation was mediated both by cell contact-dependent mechanisms and by the release of soluble factors. By adding
specific inhibitors, we identified PGE2as a soluble mediator of ESC immunoregulation. Expansion of regulatory T cells was found
in lymphoid organs and in the grafts of IVF-ESC– and NT-ESC–tolerized mice. Our study demonstrates that both IVF-ESCs and
NT-ESCs modulate recipient immune response toward tolerance to solid organ transplantation, and that NT-ESCs exhibit a lower
tendency for teratoma formation. Because NT-ESCs are obtained by NT of a somatic cell from living individuals into an
enucleated oocyte, they could represent a source of donor-derived stem cells to induce tolerance to solid organ allograft.
Journal of Immunology, 2011, 186: 4164–4174.
O
The
rgan transplantation is now firmly established as the
therapy of choice for end-stage organ failure. Improve-
ments in immunosuppressive drugs have led to out-
standing short-term patient and graft survival rates. Despite this
short-term success, the long-term kidney allograft survival has
not shown a similar rate of improvement. Chronic allograft ne-
phropathy remains the most common cause of late allograft loss,
whereas cardiovascular disease remains the leading cause of death
post-transplantation (1–6). Therefore, to improve transplantation
outcomes, it is critical to continue the development of novel
strategies to prevent acute and chronic graft rejection, whereas
lessening the need for lifelong immunosuppression, and to pro-
mote the development of a state of donor-specific tolerance.
Induction of mixed chimerism by donor-derived hematopoietic
stem cell transplantation in conjunction with myeloablative con-
ditioning has been the first and, so far, the sole successful approach
for donor-specific tolerance to organ transplantation in humans (7–
9). However, even if successful, this myeloablative protocol is so
far too difficult and complex to be broadly applicable. Efforts are
therefore under way to discover novel strategies for the induction
of tolerance possibly based on donor stem cell infusion without
the need of preconditioning procedures. In this context, embryonic
stem cells (ESCs) have been proposed as an alternative source of
donor stem cells to induce immune tolerance in the transplantation
setting (10). ESCs derived from the inner cell mass (ICM) of the
preimplantation blastocyst have the potential to generate all tis-
sues and are capable of self-renewal and unlimited proliferation
(11–13). Several reports showed that ESCs possess important
immunoprivileged properties. ESCs and their derivatives were
shown to express very low levels of MHC class I (MHC-I) Ags, to
lack expression of MHC class II (MHC-II) and costimulatory
molecules, to bypass immune recognition by natural NK cells, and
to inhibit T cell proliferation induced by third-party APCs (10,
*Department of Molecular Medicine, “Mario Negri” Institute for Pharmacological
Research, Bergamo 24125, Italy;†Transplant Research Center Chiara Cucchi De
Alessandri and Gilberto Crespi, “Mario Negri” Institute for Pharmacological Re-
search, Ranica, Bergamo24020, Italy;‡Laboratorio di Biologia dello Sviluppo, Dipar-
timento di Biologia Animale, Centro di Eccellenza in Biologia Applicata, Universita `
degli Studi di Pavia, Pavia 27100, Italy;xCentro di Ricerca Interdipartimentale di
Ingegneria Tissutale, Universita ` degli Studi di Pavia, Pavia 27100, Italy;{Sezione di
Istologia e Embriologia, Dipartimento di Medicina Sperimentale, Universita ` degli
StudidiParma,Parma43100,Italy;‖FondazioneIstitutodiRicoveroeCuraaCarattere
Scientifico Policlinico San Matteo, Pavia 27100, Italy; and#Unita ` di Nefrologia e
Dialisi, Azienda Ospedaliera, Ospedali Riuniti di Bergamo, Bergamo 24128, Italy
1B.I. and F.C. contributed equally to this work.
2Current address: Institute for Stem Cell Biology and Regenerative Medicine, Stan-
ford University School of Medicine, Palo Alto, CA.
Received for publication February 25, 2010. Accepted for publication January 27,
2011.
This work was supported by Foundation ART for Research on Transplantation Onlus
(Milano, Italy); Fondazione Internazionale Onlus Aiuti per la Ricerca sulle Malattie
Rare (Bergamo, Italy); Fondazione CARIPLO; Fondazione Istituto di Ricovero e
Cura a Carattere Scientifico Policlinico San Matteo di Pavia, Alma Mater Ticinensis;
and Roche Organ Transplantation Research Foundation (Grant 848262810).
Address correspondence and reprint requests to Federica Casiraghi at the current ad-
dress: Centro Anna Maria Astori, Parco Scientifico Tecnologico Kilometro Rosso, Via
Stezzano 87, 24126 Bergamo, Italy. E-mail address: federica.casiraghi@marionegri.it
The online version of this article contains supplemental material.
Abbreviations used in this article: AP, alkaline phosphatase; ESC, embryonic stem cell;
FasL, Fas ligand; HPF, high-power field;ICM, inner cell mass; IVF, invitrofertilization;
MHC-I,MHCclassI;MHC-II,MHCclassII;NT,nucleartransfer;Oct-4,Octamer-4;PI,
propidium iodide; RESC, rat ESC-like cell; SSEA-1, stage-specific embryonic Ag-1;
Treg, regulatory T cell.
Copyright?2011byTheAmericanAssociationofImmunologists,Inc.0022-1767/11/$16.00
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