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1 The New England Journal of Medicine Copyright 2002 by the Massachusetts Medical Society VOLUME 346 J ANUARY 3, 2002 NUMBER 1 CHIMERISM OF THE TRANSPLANTED HEART FEDERICO QUAINI, M.D., KONRAD URBANEK, M.D., ANTONIO P. BELTRAMI, M.D., NICOLETTA FINATO, M.D., CARLO A. BELTRAMI, M.D., BERNARDO NADAL-GINARD, M.D., PH.D., JAN KAJSTURA, PH.D., ANNAROSA LERI, M.D., AND PIERO ANVERSA, M.D. ABSTRACT Background Cases in which a male patient receives a heart from a female donor provide an unusual opportunity to test whether primitive cells translocate from the recipient to the graft and whether cells with the phenotypic characteristics of those of the recipient ultimately reside in the donor heart. The Y chromosome can be used to detect migrated undifferentiated cells expressing stem-cell antigens and to discriminate between primitive cells derived from the recipient and those derived from the donor. Methods We examined samples from the atria of the recipient and the atria and ventricles of the graft by fluorescence in situ hybridization to determine whether Y chromosomes were present in eight hearts from female donors implanted into male patients. Primitive cells bearing Y chromosomes that expressed c-kit, MDR1, and Sca-1 were also investigated. Results Myocytes, coronary arterioles, and capillaries that had a Y chromosome made up 7 to 10 percent of those in the donor hearts and were highly proliferative. As compared with the ventricles of control hearts, the ventricles of the transplanted hearts had markedly increased numbers of cells that were positive for c-kit, MDR1, or Sca-1. The number of primitive cells was higher in the atria of the hosts and the atria of the donor hearts than in the ventricles of the donor hearts, and 12 to 16 percent of these cells contained a Y chromosome. Undifferentiated cells were negative for markers of bone marrow origin. Progenitor cells expressing MEF2, GATA-4, and nestin (which identify the cells as myocytes) and Flk1 (which identifies the cells as endothelial cells) were identified. Conclusions Our results show a high level of cardiac chimerism caused by the migration of primitive cells from the recipient to the grafted heart. Putative stem cells and progenitor cells were identified in control myocardium and in increased numbers in transplanted hearts. (N Engl J Med 2002;346:5-15.) Copyright 2002 Massachusetts Medical Society. THE interaction between donor and recipient cells after transplantation has received great attention in an attempt to identify the basis of rejection and graft-versus-host disease. 1-3 Cell migration from the allograft to the recipient results in systemic chimerism, 1,4 and cell migration from the host to the transplanted organ results in chimerism in the organ. 2,5 Chimerism may be detected easily after sex-mismatched organ transplantation with the use of fluorescence in situ hybridization for the Y chromosome. 6-8 Systemic chimerism may be recognized when a female host receives an organ from a male donor, and chimerism may be identified in the organ after the transplantation of an organ from a female donor into a male patient. The origin and fate of recipient cells in the transplanted human heart are unknown. At present, there is no proof that chimerism leads to the generation of differentiated myocytes and intact coronary-artery branches. 9,10 Experimental evidence points to the contribution of the host s cells to neointimal thickening of intramural coronary vessels and transplant-related vasculopathy. 11,12 However, the formation of normal myocytes, arterioles, and capillaries has not been shown to occur in the grafted heart. Recent demonstrations of the ability of primitive cells to mobilize and home to the infarcted heart have raised the possibility that undifferentiated cells may translocate from the recipient to the graft, contributing to ventricular remodeling. These cells, together with circulating endothelial- and smoothmuscle cell progenitors, 16,17 could colonize the new heart. Such a form of chimerism could regenerate myocardium and sustain cardiac performance. To test this hypothesis, we studied male patients From the Department of Medicine, New York Medical College, Valhalla (F.Q., K.U., A.P.B., B.N.-G., J.K., A.L., P.A.); and the Department of Pathology, University of Udine, Udine, Italy (N.F., C.A.B.). Address reprint requests to Dr. Anversa at the Department of Medicine, Vosburgh Pavilion, New York Medical College, Valhalla, NY 10595, or at piero_anversa@nymc.edu. N Engl J Med, Vol. 346, No. 1 January 3,

2 who received hearts from female donors. Normal hearts obtained at autopsy from male and female cadavers were used to establish the efficiency and specificity, respectively, of fluorescence in situ hybridization. Three surface markers were used for the identification of primitive cells: c-kit, which is the receptor for stemcell factor 18 ; MDR1, which is a P-glycoprotein capable of extruding dyes, toxic substances, and drugs 19 ; and Sca-1, which is involved in cell signaling and cell adhesion. 20 METHODS Hearts and Detection of the Y Chromosome Eight hearts from female donors transplanted into male recipients were investigated. Permission for postmortem examination was obtained from the next of kin. Portions of the recipients atria that had been retained and sutured to the atria of the transplanted heart at the time of surgery and the atria and left ventricle of the donor hearts were sampled, fixed in formalin, and embedded in paraffin. 21 Normal hearts obtained at autopsy from six male and four female cadavers were used as controls. Four sections from each atrium of the recipient, four sections from each atrium of the donor heart, and six sections from the left ventricle of the donor heart were analyzed in each case. The Y chromosome was detected by fluorescence in situ hybridization in nuclei in interphase with the use of the DNA probe CEP Y satellite III (Vysis, Downers Grove, Ill.). 7 Nuclei were stained with propidium iodide 14 ; 16,834 nuclei were counted in myocytes, 25,642 in coronary arterioles, and 15,539 in capillaries. Cell Markers Antibodies against c-kit (Dako, Carpinteria, Calif.), MDR1 (Chemicon, Temecula, Calif.), and Sca-1 (Cedarlane, Hornby, Ont., Canada) were used to identify primitive cells. 14 Myocytes were recognized by means of antibodies against sarcomeric a-actin (Sigma, St. Louis), cardiac myosin heavy chain (Chemicon), desmin (Sigma), connexin 43 (Sigma), GATA-4 (Santa Cruz, Santa Cruz, Calif.), MEF2D (Santa Cruz), and nestin (Developmental Studies Hybridoma Bank, Iowa City, Iowa). Smooth-muscle cells were identified by means of antibodies against smooth-muscle a-actin (Sigma); fibroblasts were identified by means of antibodies against vimentin (Sigma) in the absence of factor VIII. Antibodies against Flk1 (Santa Cruz), factor VIII (Sigma), and CD31 (Santa Cruz) were used to detect endothelial cells. Antibodies against CD45, CD45RO and CD8 (Dako), and glycophorin A (Sigma) were used to detect myeloid, lymphoid, and erythroid cells, respectively. IgG antibodies conjugated with fluorescein isothiocyanate, cytochrome CY5, or tetramethylrhodamine isothiocyanate were used as secondary antibodies. 14,21 Ki-67 in nuclei was evaluated with the use of anti Ki-67 antibodies (Diagnostic Biosystems, Pleasanton, Calif.). 21 Statistical Analysis Results are presented as means ±SD. The significance of differences between two measurements was determined by Student s t-test; for multiple comparisons the Bonferroni method was used. 22 RESULTS Study Patients Data on age, primary disease, and the time from the onset of heart failure to transplantation are shown in Table 1, along with the time from transplantation to death and the weight of the implanted heart at the time of death. The female donors were a mean (±SD) of 43±15 years old and had died of cerebral hemorrhage or trauma. The donor hearts remained implanted for a period ranging from 4 to 552 days. The transplant recipients were treated with conventional immunosuppressive therapy. With one exception, only low levels of rejection (grade I) were detected. The average weight of the recipients hearts was greater than that of the donor hearts, because hearts were transplanted from female donors into diseased male patients. The smaller size of the transplanted heart and terminal cardiac failure in the recipients imposed a dramatic increase in workload on the implanted heart. TABLE 1. CLINICAL AND ANATOMICAL CHARACTERISTICS OF THE PATIENTS. INTERVAL BETWEEN CONGESTIVE HEART FAILURE AND TRANSPLANTATION WEIGHT OF DONOR HEART AT DEATH OF RECIPIENT INTERVAL BETWEEN TRANSPLANTATION AND DEATH BODY WEIGHT AGE PRIMARY DISEASE WEIGHT OF NATIVE HEART PATIENT CAUSE OF DEATH kg yr mo grams days Ischemic cardiomyopathy Graft failure Ischemic cardiomyopathy Graft failure Ischemic cardiomyopathy Sepsis Idiopathic dilated cardiomyopathy Graft failure Graft failure after previous heart transplantation Acute cardiac rejection Ischemic cardiomyopathy Sepsis Valvular disease Transplant vasculopathy Ischemic cardiomyopathy Graft failure N Engl J Med, Vol. 346, No. 1 January 3,

3 CHIMERISM OF THE TRANSPLANTED HEART Y Chromosome The left ventricular sections from six normal control hearts from male cadavers showed Y chromosomes in a mean (±SD) of 44±4 percent of myocytes (nuclei sampled, 6000; Y-chromosome positive nuclei, 2647), 50±6 percent of coronary arterioles (arterioles sampled, 587; Y-chromosome positive arterioles, 293), and 46±7 percent of capillaries (nuclei sampled, 2440; Y-chromosome positive nuclei, 1122). The prevalence of Y chromosomes in the nuclei of the vascular smooth-muscle cells of individual arterioles varied both within and among hearts and ranged from 31 percent (5 of 16 nuclei) to 75 percent (12 of 16 nuclei). The hybridization signal consisted of a single dot at the periphery of the nucleus. Four normal hearts from female cadavers were used as negative controls, and in 32 sections (8 from each heart), no myocyte nucleus, smooth-muscle cell nucleus, or endothelial-cell nucleus contained the Y chromosome. On the basis of the data collected from the examination of the hearts from male cadavers that were used as controls for evaluating the assay, our method underestimated the frequency of positive cells by nearly 50 percent. However, it was highly specific. The pattern of Y-chromosome labeling in the myocytes and coronary vessels of the hearts transplanted from female cadavers (Fig. 1A to 1H) was identical to that found in the control hearts from male cadavers. Chimerism was present in all of the transplanted hearts. The quantitative evaluation was restricted to myocytes and coronary vessels with normal structure. Areas with myointimal thickening or tissue damage were excluded from the measurements in order to avoid sites of injury in which circulating inflammatory and immunoreactive cells could have lodged. Bloodcell migration occurs in animals 2,4,5 and humans after the transplantation of a heart, a kidney, or a liver. 1 Our objective was to elucidate the role of chimerism in the undamaged myocardium. In the transplanted hearts, similar percentages of myocytes (9±4 percent), arterioles (10±3 percent), and capillaries (7±1 percent) contained the male chromosome. Arterioles were considered positive when a minimum of 30 percent of smooth-muscle cells had the Y chromosome. Often, more than 45 percent of these cells carried the Y chromosome. The fraction of male endothelial cells in the lumen of arterioles varied from 21 to 50 percent. The absence of CD45 on the surface of these cells indicated that they were not inflammatory infiltrates. The 50 percent efficiency of fluorescence in situ hybridization for the Y chromosome implied that at least 60 percent of smooth-muscle cells and 42 percent of endothelial cells in the arteriolar wall were of male origin. The high level of chimerism in arterioles was consistent with the formation of resistance vessels in the recipient. Cells from the host were responsible for the development of 14 percent of the capillaries (Fig. 1H). Because of the small number of patients, we could not study the correlation between the time from transplantation to death and the level of chimerism present in myocytes, arterioles, and capillaries. However, the highest levels of chimerism in myocytes (15 percent), arterioles (12 percent), and capillaries (9 percent) were found between 4 and 28 days after transplantation. Conversely, the lowest levels of chimerism in myocytes (4 percent), arterioles (7 percent), and capillaries (5 percent) were noted between 396 and 552 days after transplantation. Most Y-chromosome bearing cells were fully mature and indistinguishable from adjacent and distant negative cells. Occasionally, small myocytes were observed. Cell proliferation was measured with the use of Ki-67 labeling combined with Y-chromosome labeling (Fig. 1I, 1J, and 1K). Nine percent of myocytes contained the Y chromosome, and a mean of 17.2±4.2 percent of this group of cells were replicating (nuclei counted, 862). In contrast, only 1.0±0.3 percent of the remaining 91 percent of myocytes were replicating. Similarly, 13.6±4.8 percent of the 10 percent of smooth-muscle cells that were male (nuclei counted, 1165) and 16.0±4.9 percent of the 7 percent of endothelial cells that were male (nuclei counted, 1141) were replicating. Of the remaining 90 percent of smoothmuscle cells and 93 percent of endothelial cells, 0.8± 0.2 percent and 1.1±0.3 percent, respectively, were replicating. Y-chromosome positive mitotic myocytes, endothelial cells, and smooth-muscle cells were found. Primitive Cells and the Transplanted Heart Another objective of this study concerned the origin of male cells that translocated and differentiated in hearts transplanted from female donors. In six cases, during cardiac transplantation, portions of both atria of the recipient were sutured to the partially dissected atria of the donor. In the other two cases, only the left atrium of the recipient was maintained, since, on the right side, an anastomosis was performed between the vena cava of the recipient and that of the donor heart. The presence of hybrid atria raised the question of whether undifferentiated cells migrated from the host to the graft through the systemic circulation or homed to the ventricles from the native atrial tissue that had been preserved. Circulating primitive cells were not evaluated. However, primitive cells in the atria of the recipient and the atria and left ventricle of the donor were measured after they had been identified by means of c-kit, MDR1, and Sca-1. These surface proteins are present in stem cells but are not exclusive to this type of cell. 18,23-26 Cells expressing c-kit, MDR1, or Sca-1 (Fig. 2A, 2B, and 2C) were identified in the atria and left ventricle. These were small, round cells with a large nu- N Engl J Med, Vol. 346, No. 1 January 3,

4 A E B F C G D H 8 N Engl J Med, Vol. 346, No. 1 January 3,

5 CHIMERISM OF THE TRANSPLANTED HEART I J K Figure 1. The Y Chromosome in Transplanted Hearts. The arrowheads indicate the Y chromosomes in myocytes (Panels A and B), smooth-muscle cells (Panels C and D), and endothelial cells in both coronary arterioles (Panels E and F), and capillary endothelial cells (Panels G and H), and in the nuclei of a myocyte (Panel I), a smooth-muscle cell (Panel J), and a capillary endothelial cell (Panel K). In Panels A through H, the blue areas show the propidium iodide staining in nuclei, and the green areas indicate the Y chromosomes in nuclei. The red areas indicate the presence of sarcomeric a-actin in Panel B, of smooth-muscle a-actin in Panel D, and of factor VIII in Panels F and H. In Panels I, J, and K, the bright blue, fluorescent areas and the arrows indicate the presence of Ki-67, and the yellow areas show the Y chromosomes. The scale bars represent 10 µm. cleus and a thin rim of cytoplasm. The colocalization of c-kit and MDR1 in these cells was also documented (Fig. 2D, 2E, and 2F). Sca-1 was not found in cells that contained c-kit or MDR1. The undifferentiated cells were negative for markers of bone marrow derived cells, such as leukocyte common antigen (CD45), lymphoid lineage (CD45RO and CD8), and erythroid progeny (glycophorin A) (Fig. 2J, 2K, 2L, 2M, and 2N). These cardiac cells were negative for markers of differentiated myocytes (cardiac myosin heavy chain, sarcomeric a-actin, desmin, and connexin 43), endothelial cells (CD31, factor VIII, and vimentin), smooth-muscle cells (smooth-muscle a-actin and desmin), and fibroblasts (vimentin). In addition, fluorescence in situ hybridization assays for the Y chromosome were evaluated in these primitive cells in samples from the atria and left ventricle of the donor (Fig. 2G, 2H, and 2I). The 10 left ventricles from the control hearts had low numbers of cells that were positive for c-kit, MDR1, or Sca-1 (Fig. 3). In the left ventricles of the eight hearts transplanted from female donors, the number of cells expressing c-kit was 4.0 times as high as that in the control hearts (P<0.001); the number of cells expressing MDR1 was 3.9 times as high (P< 0.001); and the number of cells expressing Sca-1 was 6.0 times as high (P<0.001). Values for the residual atrial portion of the recipients were similar to those for the atria of the donor hearts but were much higher than those for the left ventricle of the donor hearts (Fig. 3). The prevalence of primitive cells expressing c-kit was 1.3 times as high in the atria of the donor hearts as in the ventricle of the donor hearts (P= 0.006); the prevalence of primitive cells expressing MDR1 was 2.4 times as high (P<0.001); and the prevalence of primitive cells expressing Sca-1 was 2.6 times as high (P<0.001) (Fig. 3). In the atria and ventricles of the donor hearts, 12 to 16 percent of the cells positive for c-kit, MDR1, or Sca-1 contained the Y chromosome. In donor and recipient myocardium, 29 to 40 percent of the c-kit positive cells also expressed MDR1. Similarly, 14 to 18 percent of MDR1- positive cells also expressed c-kit. Chimerism and Amplifying Cardiac Cells To identify the cells involved in the generation of myocytes and vessels of host origin in the heart transplanted from a female donor, early markers of cardiaccell lineages were identified. The transcription factors MEF2D and GATA-4 were recognized in Y-chromosome bearing cells (Fig. 4A and 4B), documenting that these cells were committed to myocyte differentiation. Moreover, Flk1 receptor was detected (Fig. 4C), suggesting the involvement of endothelial and smooth-muscle cell lineages. The intermediate filament protein nestin was also observed (Fig. 4D), implying N Engl J Med, Vol. 346, No. 1 January 3,

6 A D B E C F Figure 2. Primitive Cells Expressing MDR1, c-kit, and Sca-1 in Transplanted Hearts and the Native Atria of Recipients. Panels A, B, and C show the native atrium of a transplant recipent; the arrowheads indicate the presence of MDR1 in primitive cells; R-A denotes the direction of the recipient atrium, and D-A the direction of the donor atrium. Panels D, E, and F show c-kit and MDR1 in a primitive cell in the atrium of a donor heart. The blue areas in Panels A through F show the propidium iodide staining in nuclei; the green areas in Panels B, C, E, and F indicate the presence of MDR1; the yellow areas in Panels D and F indicate the presence of c-kit; and the red areas in Panels C and F indicate the presence of sarcomeric a-actin. Panels G, H, and I show Y chromosomes (light green, arrowheads) in an atrial cell expressing c-kit (Panel G, red, arrow), a ventricular cell expressing MDR1 (Panel H, red, arrow), and a ventricular cell expressing Sca-1 (Panel I, red, arrow); the green areas indicate the presence of sarcomeric a-actin. In Panel J, a cell expressing c-kit (green, arrow) is negative for CD45 (red, arrowheads); in Panel K, a cell expressing MDR1 (green, arrow) is negative for CD45 (red, arrowheads); in Panel L, a cell expressing c-kit (green, arrow) is negative for glycophorin A (red, arrowheads); in Panel M, a cell expressing MDR1 (green, arrow) is negative for CD45RO (red, arrowheads); and in Panel N, a cell expressing Sca-1 (green, arrow) is negative for CD8 (red, arrowheads). The scale bars represent 10 µm. 10 N Engl J Med, Vol. 346, No. 1 January 3,

7 CHIMERISM OF THE TRANSPLANTED HEART G K H L I M J N N Engl J Med, Vol. 346, No. 1 January 3,

8 Cells per 100 mm 2 Cells per 100 mm 2 Cells per 100 mm c-kit MDR1 Sca-1 Control Donor Left Ventricle Recipient Atria Donor Figure 3. Numbers of Primitive Cells Expressing c-kit, MDR1, and Sca-1 in the Left Ventricle of Transplanted Hearts and Control Hearts and in the Atria of Transplanted Hearts and Transplant Recipients. The numbers of cells expressing c-kit, MDR1, and Sca-1 counted in the native atria of recipients were 633, 971, and 481, respectively. Values for the atria of the donor hearts were 647, 1233, and 556, respectively; values for the left ventricle of the donor hearts were 163, 405, and 178, respectively; and values for the left ventricle of the control hearts were 48, 55, and 24, respectively. P<0.05 for all comparisons with the control hearts and for all comparisons with the left ventricle of the donor hearts. a more advanced stage of myocyte differentiation. 27 Although Figure 4 provides examples of the presence of these proteins in Y-chromosome positive cells, the majority of cardiac cells with these markers had negative results on fluorescence in situ hybridization. These indicators of cell differentiation were not seen in primitive cells that expressed only c-kit, MDR1, or Sca-1. DISCUSSION We report here that undifferentiated cells were found in control human hearts and that their number increased significantly in hearts from female donors that were transplanted into male recipients. These primitive cells expressed on their surface stem-cell related antigens including c-kit, MDR1, and Sca A fraction of these cells were Y-chromosome positive, providing direct evidence of their origin: they had translocated from the host to the atria and ventricles of the grafted heart. Loss of stem-cell markers, active proliferation, and acquisition of the mature phenotype followed the cell colonization. New myocytes, coronary arterioles, and capillaries were formed. After large infarcts, lineage-negative, c-kit positive, 14 CD34-positive 13 and highly enriched hematopoietic stem cells (side population) of the bone marrow 15 migrate to damaged areas and promote repair. Although tissue injury occurs with transplantation, 9,12,28 we observed that myocytes and coronary vessels were generated within the intact myocardium. Through growth and differentiation, male primitive cells contributed to the remodeling of the heart transplanted from a female donor. This conclusion is consistent with the determination that 18 percent of myocytes, 20 percent of coronary arterioles, and 14 percent of capillaries were of male origin, according to the values that result when the 50 percent efficiency of the fluorescence in situ hybridization assay is taken into account. The source of primitive cells that lead to cardiac chimerism is difficult to identify. Circulating hematopoietic stem cells from the recipient could have homed to the implanted heart. 13,15 Early indicators of bone marrow cell differentiation were not detected in cells expressing c-kit, MDR1, or Sca-1, whether or not they had the Y chromosome. However, these findings do not preclude the possibility that stem cells were mobilized from the bone marrow and reached the implanted heart. In the graft, a high number of undifferentiated cells were Y-chromosome negative, suggesting that groups of primitive cells reside in the heart and, together with the cells translocated from the host, multiply and acquire cardiac-cell lineages. At present, it is impossible to establish whether replicating female cells originate from stem cells or derive from subpopulations of nonterminally differentiated cells. Chimerism in transplanted organs has been linked to the process of rejection. 1,2,28 It has been claimed 12 N Engl J Med, Vol. 346, No. 1 January 3,

9 CHIMERISM OF THE TRANSPLANTED HEART A C B D Figure 4. MEF2D (Panel A), GATA-4 (Panel B), Flk1 (Panel C), and Nestin (Panel D) in Committed Cells (Red, Arrows) Containing the Y Chromosome (Yellow, Arrowheads). The blue areas show propidium iodide staining in the nuclei; the green areas indicate the presence of sarcomeric a-actin. The scale bars represent 10 µm. that cell death, inflammatory infiltrates, and the release of cytokines characterize the immunoreactive response Humoral factors may act as molecular signals for the chemoattraction and activation of quiescent primitive cells. Cardiac chimerism was not previously identified in humans, because this phenomenon was considered to be restricted to hemolymphopoietic cells. 1,2,28 Our results contrast with previous observations. 9,10 More refined techniques and the use of confocal microscopy with enhanced resolution 14 have improved the analysis of the myocardium. We can only speculate as to the pathobiologic sequence of events. When it is transplanted, the donor heart has to reverse the clinical manifestations of endstage heart failure in the recipient, 32,33 including an increased hemodynamic load. These mechanical factors most likely stretch the myocardium while triggering the translocation of undifferentiated cells clustered in the host s native atrium and concurrently activate resident cells in the transplanted heart. Locally distributed primitive cells and those that have migrated from the systemic circulation may contribute to optimizing cardiac mass and restoring function in the short term. Severe depression in ventricular performance with the progression of coronary vasculopathy and tissue damage may sustain over the long term the growthpromoting effects of native and colonizing primitive cells in the transplanted heart. The high degree of differentiation of the myocytes, coronary arterioles, and capillaries that originated from male cells and were present in the transplanted heart suggests that cell migration occurred early and involved primitive cells (i.e., stem cells) and precursor cells (i.e., committed progenitors). Precursor cells proliferate much more rapidly than primitive cells, undergo differentiation, and acquire functional compe- N Engl J Med, Vol. 346, No. 1 January 3,

10 tence. 34 Y-chromosome positive cells from the graft whose host survived only four days after transplantation were indistinguishable from those of transplants of longer duration and from the host s cells. Because the earliest migration date of these cells was the date of transplantation, their mature phenotype indicates that the migration of primitive cells into the transplanted heart, cell differentiation, and phenotypic maturation were rapid processes. This temporal sequence is more reminiscent of organ morphogenesis and cell differentiation during embryonic and fetal development than of the rate of organ remodeling expected in an adult. The identification of male progenitor cells expressing MEF2D, GATA-4, nestin, and Flk1 supports this contention. An important question concerns whether, at the completion of differentiation, each cardiac-cell lineage reaches an arrest of growth so that the ability to replicate is permanently lost. Endothelial and smooth-muscle cells continue to grow in vitro 35,36 and in vivo. 36,37 Mitotic division and cell regeneration of myocytes also occur in vivo in the adult heart in animals and humans, 21,38 but mature myocytes do not proliferate in vitro. 39 Thus, it seems that there are resident cardiac stem cells in vivo that differentiate into myocytes in normal and diseased hearts. These cells are not confined to restricted regions of the heart; they migrate where they are needed, as demonstrated by the high level of cardiac chimerism found in this study. Supported by grants (HL-38132, HL-39902, AG-15756, HL-65577, HL-66923, HL-65573, and AG-17042) from the National Institutes of Health. The monoclonal antibody Rat-401 (anti-nestin) developed by Hockfield was obtained from the Developmental Studies Hybridoma Bank at the Department of Biological Sciences, University of Iowa, Iowa City, operating under the auspices of the National Institute of Child Health and Human Development. 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