Survival and maturation of human embryonic stem cell-derived cardiomyocytes in rat hearts.
Human embryonic stem cell (hESC)-derived cardiomyocytes are a promising cell source for cardiac repair. Whether these cells can be transported long distance, survive, and mature in hearts subjected to ischemia/reperfusion with minimal infarction is unknown. Taking advantage of a constitutively GFP-expressing hESC line we investigated whether hESC-derived cardiomyocytes could be shipped and subsequently form grafts when transplanted into the left ventricular wall of athymic nude rats subjected to ischemia/reperfusion with minimal infarction. Co-localization of GFP-epifluorescence and cardiomyocyte-specific marker staining was utilized to analyze hESC-derived cardiomyocyte fate in a rat ischemia/reperfused myocardium. Differentiated, constitutively green fluorescent protein (GFP)-expressing hESCs (hES3-GFP; Envy) containing about 13% cardiomyocytes were differentiated in Singapore, and shipped in culture medium at 4 degrees C to Los Angeles (shipping time approximately 3 days). The cells were dissociated and a cell suspension (2 x 10(6) cells for each rat, n=10) or medium (n=10) was injected directly into the myocardium within the ischemic risk area 5 min after left coronary artery occlusion in athymic nude rats. After 15 min of ischemia, the coronary artery was reperfused. The hearts were harvested at various time points later and processed for histology, immunohistochemical staining, and fluorescence microscopy. In order to assess whether the hESC-derived cardiomyocytes might evade immune surveillance, 2 x 10(6) cells were injected into immune competent Sprague-Dawley rat hearts (n=2), and the hearts were harvested at 4 weeks after cell injection and examined as in the previous procedures. Even following 3 days of shipping, the hESC-derived cardiomyocytes within embryoid bodies (EBs) showed active and rhythmic contraction after incubation in the presence of 5% CO(2) at 37 degrees C. In the nude rats, following cell implantation, H&E, immunohistochemical staining and GFP epifluorescence demonstrated grafts in 9 out of 10 hearts. Cells that demonstrated GFP epifluorescence also stained positive (co-localized) for the muscle marker alpha-actinin and exhibited cross striations (sarcomeres). Furthermore, cells that stained positive for the antibody to GFP (immunohistochemistry) also stained positive for the muscle marker sarcomeric actin and demonstrated cross striations. At 4 weeks engrafted hESCs expressed connexin 43, suggesting the presence of nascent gap junctions between donor and host cells. No evidence of rejection was observed in nude rats as determined by inspection for lymphocytic infiltrate and/or giant cells. In contrast, hESC-derived cardiomyocytes injected into immune competent Sprague-Dawley rats resulted in an overt lymphocytic infiltrate. hESCs-derived cardiomyocytes can survive several days of shipping. Grafted cells survived up to 4 weeks after transplantation in hearts of nude rats subjected to ischemia/reperfusion with minimal infarction. They continued to express cardiac muscle markers and exhibit sarcomeric structure and they were well interspersed with the endogenous myocardium. However, hESC-derived cells did not escape immune surveillance in the xenograft setting in that they elicited a rejection phenomenon in immune competent rats.
Dai
Wangde
W
The Heart Institute, Good Samaritan Hospital, University of Southern California, 1225 Wilshire Boulevard, Los Angeles, CA 90017-2395, USA.
Field
Loren J
LJ
Rubart
Michael
M
Reuter
Sean
S
Hale
Sharon L
SL
Zweigerdt
Robert
R
Graichen
Ralph E
RE
Kay
Gregory L
GL
Jyrala
Aarne J
AJ
Colman
Alan
A
Davidson
Bruce P
BP
Pera
Martin
M
Kloner
Robert A
RA
D000818:Animals; D002454:Cell Differentiation; D002470:Cell Survival; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000637:transplantation*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D017202:Myocardial Ischemia / Q000473:pathology / Q000628:therapy*; D009206:Myocardium / Q000166:cytology* / Q000473:pathology; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism; D051381:Rats; D011923:Rats, Nude; D033581:Stem Cell Transplantation; D014183:Transplantation, Heterologous
D018031:Connexin 43; D049452:Green Fluorescent Proteins
Nature. 2006 Apr 27;440(7088):1199-203
16565704
FASEB J. 2007 May;21(7):1345-57
17284483
EMBO J. 2000 Oct 2;19(19):5060-70
11013209
FASEB J. 2000 Dec;14(15):2540-8
11099473
J Clin Invest. 2001 Aug;108(3):407-14
11489934
Circulation. 2002 Apr 9;105(14):1720-6
11940553
J Mol Med (Berl). 2002 Jun;80(6):343-50
12072909
Curr Opin Immunol. 2002 Oct;14(5):601-5
12183159
Circ Res. 2002 Sep 20;91(6):501-8
12242268
J Am Coll Cardiol. 2003 Mar 5;41(5):879-88
12628737
Circ Res. 2003 Apr 4;92(6):651-8
12600875
J Am Coll Cardiol. 2003 Apr 2;41(7):1078-83
12679204
Am J Physiol Cell Physiol. 2003 Jun;284(6):C1654-68
12584115
Cardiovasc Res. 2003 May 1;58(2):336-50
12757868
Circulation. 2003 Jun 3;107(21):2733-40
12742992
J Mol Cell Cardiol. 2003 Jun;35(6):661-70
12788384
Proc Natl Acad Sci U S A. 2003 Jun 24;100(13):7808-11
12805561
Circ Res. 2003 Jul 11;93(1):32-9
12791707
Cytotherapy. 2003;5(5):399-413
14578102
Am J Physiol Heart Circ Physiol. 2003 Dec;285(6):H2355-63
14613910
Stem Cells. 2004;22(4):448-56
15277692
J Physiol. 2004 Sep 1;559(Pt 2):479-96
15243138
J Clin Invest. 2004 Sep;114(6):775-83
15372101
Nat Biotechnol. 2004 Oct;22(10):1282-9
15448703
Differentiation. 1991 Feb;46(1):51-60
1710586
J Clin Invest. 1993 Sep;92(3):1548-54
8376605
Science. 1994 Apr 1;264(5155):98-101
8140423
J Clin Invest. 1996 Jul 1;98(1):216-24
8690796
Science. 1998 Nov 6;282(5391):1145-7
9804556
Circ Res. 2004 Dec 10;95(12):1154-66
15591237
Congest Heart Fail. 2004 Nov-Dec;10(6):293-301
15591844
Circulation. 2005 Jan 4;111(1):11-20
15611367
Circ Res. 2005 Jan 7;96(1):110-8
15576649
Nat Methods. 2005 Apr;2(4):259-60
15782217
Stem Cells. 2005 Jun-Jul;23(6):772-80
15917473
Cardiovasc Res. 2005 Aug 1;67(2):301-7
15907822
Circulation. 2005 Jul 12;112(2):214-23
15998673
Am J Pathol. 2005 Sep;167(3):663-71
16127147
Circulation. 2005 Aug 30;112(9 Suppl):I166-72
16159810
Lancet. 2005 Sep 17-23;366(9490):1005-12
16168783
Circulation. 2005 Sep 20;112(12):1748-55
16172284
Biotechnol Bioeng. 2005 Dec 30;92(7):920-33
16189818
Handb Exp Pharmacol. 2006;(174):73-100
16370325
Transpl Immunol. 2005 Dec;15(2):91-7
16412954
Annu Rev Physiol. 2006;68:29-49
16460265
Stem Cells. 2006 Feb;24(2):221-9
16109762
FASEB J. 2006 Apr;20(6):661-9
16581974
N Engl J Med. 2006 Sep 21;355(12):1274-7
16990391
J Exp Med. 2006 Oct 2;203(10):2315-27
16954371
Dev Cell. 2006 Nov;11(5):723-32
17084363
Reprod Biomed Online. 2006 Nov;13(5):712-24
17169186
Stem Cells. 2007 Jan;25(1):236-44
16973829
Stem Cells. 2006 Aug;24(8):1956-67
16675594