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European Journal of Heart Failure 2004 6(4):399-402; doi:10.1016/j.ejheart.2003.12.006
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© 2004 European Society of Cardiology

Cardiac chimerism in recipients of peripheral-blood and bone marrow stem cells

Antoni Bayes-Genisa,*, Eduardo Muñiz-Diazb, Lluis Catasusc, Marina Arillab, Carmen Rodriguezc, Jordi Sierrad, Pedro J. Madozb and Juan Cincaa

a Cardiology Service, Hospital de la Santa Creu i Sant Pau C/Sant Antoni Ma Claret 167, 08025 Barcelona, Spain
b Blood Bank, Hospital de la Santa Creu i Sant Pau C/Sant Antoni Ma Claret 167, 08025 Barcelona, Spain
c Pathology Service, Hospital de la Santa Creu i Sant Pau C/Sant Antoni Ma Claret 167, 08025 Barcelona, Spain
d Hematology Service, Hospital de la Santa Creu i Sant Pau C/Sant Antoni Ma Claret 167, 08025 Barcelona, Spain

* Corresponding author. Tel: +93-556-92-58; fax: +93-291-94-24. E-mail address: abayesgenis{at}hsp.santpau.es


   Abstract
Top
 Abstract
1. Introduction
 2. Patients and methods
 3. Results and discussion
 References
 
Multipotent progenitor cells have the ability to differentiate into most somatic cell types, including cardiac myocytes. We sought to investigate cardiac chimerism after peripheral-blood and bone marrow stem cell transplantation. Between 10 and 17 highly polymorphic short tandem repeat (STR) markers were assayed in DNA obtained from donors’ peripheral blood, recipients’ peripheral blood before transplantation, and the recipient's heart in every patient. Gender and non-gender STR donor alleles were identified in the recipient heart in three patients. Using a highly sensitive PCR assay to determine donor and recipient genotypes, we confirmed the existence of cardiac chimerism in recipients of peripheral-blood and bone marrow stem cells.

Received July 11, 2003; Revised October 27, 2003; Accepted December 22, 2003


   1. Introduction
Top
 Abstract
 1. Introduction
2. Patients and methods
 3. Results and discussion
 References
 
Multipotent progenitor cells have the ability to differentiate into most somatic cell types, including cardiac myocytes [1,2]. In humans, the ability of multipotent progenitor cells to differentiate into cardiomyocytes has been explored in allografted female hearts into male recipients, examining the heart at the time of explantation for the presence of Y chromosome-positive cells. Diverse results on the extent of cardiac chimerism after transplantation [3–5] have been obtained, but the data support the ability of the human heart of regenerating by recruiting multipotent adult cells of non-cardiac origin.

Recent reports have identified human bone marrow as a source of extra-cardiac progenitor cells capable of de novo cardiomyocyte formation [6,7]. Again, a marked discrepancy in the level of bone marrow-derived cardiomyocytes has been reported, ranging from 6.4 [6] to 0.2% [7], by means of fluorescence in situ hybridization (FISH). Moreover, only gender genes were studied in these reports, and none used confocal microscopy, the method of choice for the study of small nuclear bodies [8].

We sought to investigate the presence of cardiac chimerism after peripheral-blood and bone marrow stem cell transplantation by means of PCR of highly polymorphic short tandem repeat (STR) markers. We analyzed donor and recipient genotypes, and the presence of donor alleles in the recipient's heart.


   2. Patients and methods
Top
 Abstract
 1. Introduction
 2. Patients and methods
3. Results and discussion
 References
 
2.1. Patients
Six patients with myelogenous leukemia (three chronic and three acute) received a transplant of allogeneic peripheral-blood stem cells (four patients) or bone marrow transplantation (in two patients) (Table 1). All patients received a gender-mismatch allograft; patients 1–4 were male and received stem cells of female origin, and patients 5–6 were female and received stem cells of male origin. All allografts were from patient's siblings. The peripheral-blood stem cells were obtained by apheresis after the donor had been treated for 5 days with granulocyte-colony stimulating factor (G-CSF) at a dose of 10 µg/kg of body weight per day. The total number of CD34+ cells transplanted ranged from 8x106 kg–1 of body weight to 19x106 kg–1 of body weight.


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  		Table 1 Clinical data and genotype analysis of hematopoietic stem cells recipients

 
2.2. Methods
Total DNA from donors and recipient patients was isolated from peripheral blood using a commercial kit (Wizard, Promega Corp. Madison WI, USA). Left ventricle free wall post-transplantation specimens were retrieved from the autopsy files. Genomic DNA of myocardial samples was extracted by standard methods from formalin-fixed, paraffin-embedded tissue. Before isolation of myocardial DNA, microdissection by hand was performed to remove epicardial arteries and fat. Extreme caution was observed in the preparation of myocardial samples for subsequent PCR amplification to avoid contamination by male DNA.

To determine whether the studied samples were of donor or recipient origin a PCR assay analyzing highly polymorphic STR markers was used. We used a commercially available multiplex PCR kit (AmP FISTR Profiler Plus PCR Amplification Kit, Applied Biosystems Foster City, CA USA) with nine STR loci and the amelogenin locus for gender identification, and seven additional STR markers. A minimum of 10 polymorphisms were assayed for every patient (Table 1). One primer of each marker was labeled with different NHS-ester fluorescent dyes, which were detected on the ABI PRISM 310 Genetic Analyzer (Applied Biosystems Foster City, CA USA). Samples were scored using Genotyper and Genescan (ABI) programs.

To discriminate gender alleles in female patients who received male progenitor cells a PCR assay was performed as described elsewhere [9].


   3. Results and discussion
Top
 Abstract
 1. Introduction
 2. Patients and methods
 3. Results and discussion
References
 
The clinical characteristics of the patients who received peripheral-blood or bone marrow stem cells are shown in the Table 1. Microscopic examination of the cardiac sections showed no evidence of myocardial inflammation (not shown). We obtained a myocardial cell lysate (after surgically removing epicardial vessels and surrounding adipose tissue) for PCR testing, instead of using FISH, the method used in previous reports to demonstrate cardiac chimerism after bone marrow transplantation [6,7]. The signals obtained after PCR are positively evaluated, only the existence and not the absence of a signal is considered for evaluation. In addition, the PCR signals are directly evaluated the results obtained do not need to be corrected by certain factors to level out false-negative and false-positive results.

Between 10 and 17 highly polymorphic STR markers were assayed in DNA obtained from donor's peripheral blood, recipient's peripheral blood before transplantation, and the recipient's heart in every patient. Since all grafts were allogeneic but derived from siblings, a limited number of alleles, ranging from 1 to 6, were different between donor and recipient (Table 1). In three patients we identified cardiac chimerism. The donor genotype identified in the recipient's heart in two of these cases, female patients who received male progenitor cells, were gender alleles (Fig. 1 A and B). Non-gender STR donor alleles within the myocardium were found in a male patient recipient of female bone marrow stem cells. Fig. 1C shows in the upper row the genotype of donor and recipient, middle row shows the PCR product of the recipient myocardial cell lysate (red line) exhibiting donor alleles (green line), and lower row shows recipient's alleles (blue line) in the recipient's heart (red line).


Figure 1
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  		Fig. 1 Identification of cardiac chimerism after peripheral-blood and bone-marrow transplantation. (A) Identification of X and Y alleles in the myocardial sample of a female who received a bone marrow transplant from a male (patient #5). (B) Asterisk highlights the Y chromosome band, identified in the cardiac tissue of a female who received a peripheral-blood stem cell transplant from a male (patient # 6); female, female control DNA; male, male control DNA. (C) Upper row shows different donor (green line) and recipient (blue line) genotypes; middle row shows the PCR product of the recipient myocardial cell lysate (red line) exhibiting donor alleles (alleles 2 and 3); and lower row shows recipient's alleles in the recipient's heart (alleles 1 and 2).

 
We report the presence of cardiac chimerism in recipients of peripheral-blood and bone marrow stem cells, using a highly sensitive PCR assay for donor and recipient genotyping. However, the lack of chimerism seen at early stages after transplantation (patients 2, 3, and 4) in our present study, suggest a long-term recruitment of progenitor cells rather than an initial seeding event early after transplantation. The existence of a population of stem cells that circulate for long periods after transplantation, allowing an equilibrium to be established between circulating and tissue-specific seeding compartments, is well-known. It is also possible that stem cells that are committed to differentiation primarily along a particular pathway can switch to another lineage over time under the influence of signals of the local microenvironment [10]. A recent study postulated that circulating stem cells differentiate into dividing myocytes that repair necrotic myocardium after infarction in humans [11]. The intensity of cardiac chimerism in the normal heart is not completely defined. In hearts obtained after cardiac transplantation, chimerism ranged between 0.02±0.02% in the study by Laflamme et al. [4] and 9±4% as reported by Quaini et al. [3].

A limitation of the present study is the use of formalin-fixed, paraffin-embedded myocardial samples, which is the way our pathology department routinely stores samples after autopsy. Retrieval of DNA from paraffin-embedded samples yields fragmented pieces of DNA, hampering subsequent PCR amplification of high molecular weight STRs (>250 bp). Moreover, the exact nature of the cells identified by PCR remains unknown. They could be cardiomyocytes, but it is not excluded that endothelial or fibroblast-like cells are also recruited by the heart. Whichever cell type it is, our study shows cross-talk and homing of circulating progenitor cells into the heart.

The existence of cardiac chimerism after stem cell transplantation raises the possibility that circulating or bone marrow progenitor cells can be isolated from a patient, and delivered in a therapeutic context to support the patient's diseased heart [12]. Obtaining sufficient own progenitor cells for clinical purposes would reduce or abolish the need of strategies to counter immune rejection. Further insight is required to better understand the process of phenotypical and functional cell differentiation in the cardiac microenvironment.


   References
Top
 Abstract
 1. Introduction
 2. Patients and methods
 3. Results and discussion
 References
 

  1. Jiang Y., Jahagirdar B.N., Reinhardt R.L., et al. Pluripotency of mesenchymal stem cells derived from adult bone marrow. Nature (2002) 418:41–49.[CrossRef][Medline]
  2. Toma C., Pittenger M.F., Cahill K.S., Byrne B.J., Kessler P.D. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation (2002) 105:93–98.[Abstract/Free Full Text]
  3. Quaini F., Urbanek K., Beltrami A.P., et al. Chimerism of the transplanted heart. N Engl J Med (2002) 346:5–15.[Abstract/Free Full Text]
  4. Laflamme M.A., Myerson D., Saffitz J.E., Murry C.E. Evidence for cardiomyocyte repopulation by extracardiac progenitors in transplanted human hearts. Circ Res (2002) 90:634–640.[Abstract/Free Full Text]
  5. Bayes-Genis A., Salido M., Solé Ristol F., et al. Host-cell derived cardiomyocytes in sex-mismatch cardiac allografts. Cardiovasc Res (2002) 56:404–410.[Abstract/Free Full Text]
  6. Thiele J., Varus E., Wickenhauser C., Kvasnicka H.M., Metz K., Schaefer H.W., Beelen D.W. Chimerism of cardiomyocytes and endothelial cells after allogeneic bone marrow transplantation in chronic myeloid leukemia: an autopsy study. Pathologe (2002) 23:405–410.[CrossRef][Web of Science][Medline]
  7. Deb A., Wang S., Skelding K.A., Miller D., Simper D., Caplice N.M. Bone marrow-derived cardiomyocytes are present in adult human heart. Circulation (2003) 107:1247–1249.[Abstract/Free Full Text]
  8. Anversa P., Nadal-Ginart B. Cardiac chimerism: methods matter. Circulation (2002) 106:e129–e131.[CrossRef][Web of Science][Medline]
  9. Kogan S.C., Gitschier J. Genetic prediction of hemophilia A. In: PCR protocols: a guide to methods and applications—Innis M.A., Gelfand D.H., Sninsky J.J., White T.J., eds. (1990) San Diego: Academic Press. 288–299.
  10. Korbling M., Katz R.L., Khanna A., et al. Hepatocytes and epithelial cells of donor origin in recipients of peripheral-blood stem cells. N Engl J Med (2002) 346:738–746.[Abstract/Free Full Text]
  11. Beltrami A.P., Urbanek K., Kajstura J., et al. Evidence that human cardiac myocytes divide after myocardial infarction. N Engl J Med (2001) 344:1750–1757.[Abstract/Free Full Text]
  12. Strauer B.E., Brehm M., Zeus T., et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation (2002) 106:1913–1918.[Abstract/Free Full Text]

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