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(The FASEB Journal. 2006;20:950-952.)
© 2006 FASEB

Purified human hematopoietic stem cells contribute to the generation of cardiomyocytes through cell fusion

Fumihiko Ishikawa*,¶,1,2, Hideki Shimazu*,1, Leonard D. Shultz||, Mitsuhiro Fukata*, Ryu Nakamura{ddagger}, Bonnie Lyons||, Kazuya Shimoda*, Shinji Shimoda*, Takaaki Kanemaru*, Kei-ichiro Nakamura{dagger}, Hiroyuki Ito*, Yoshikazu Kaji*, Anthony C.F. Perry§ and Mine Harada*

* Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan;

{dagger} Second Department of Anatomy, Kurume University School of Medicine, Kurume, Japan;

{ddagger} Zeiss Japan, Tokyo, Japan;

§ Laboratory of Mammalian Molecular Embryology, RIKEN Center for Developmental Biology, Kobe, Japan;

|| The Jackson Laboratory, Bar Harbor, Maine; and

Research Unit for Human Disease Model, RIKEN Center for Allergy and Immunology, Yokohama, Japan

2Correspondence: Kyushu University Graduate School of Medical Sciences, Department of Medicine and Biosystemic Science, 3–1-1 Maidashi, Higashi-ku, Fukuoka 812-8582. E-mail: f_ishika{at}intmed1.med.kyushu-u.ac.jp

ABSTRACT

To obtain insights into the cardiomyogenic potential of hematopoietic tissue, we intravenously (i.v.) injected purified hematopoietic stem/progenitor cells into newborn recipients that may fully potentiate the developmental plasticity of stem cells. Transplantation of mouse bone marrow (BM) lineage antigen-negative (Lin–) cells resulted in the generation of the cells that displayed cardiomyocyte-specific antigenic profiles and contractile function when transplanted into syngeneic newborn recipients. To clarify the mechanism underlying the cardiomyogenic potential, green fluorescent protein (GFP)-labeled BM Lin–ScaI+ hematopoietic progenitors were transplanted into neonatal mice constitutively expressing cyan fluorescence protein (CFP). Lambda image acquisition and linear unmixing analysis using confocal microscopy successfully separated GFP and CFP, and revealed that donor GFP+ cardiomyocytes coexpressed host-derived CFP. We further reconstituted human hemopoietic- and immune systems in mice by injecting human cord blood (CB)-derived Lin–CD34+CD38– hematopoietic stem cells (HSCs) into neonatal T cell–B cell–NK cell– immune-deficient NOD/SCID/IL2r{gamma}null mice. Fluoroescence in situ hybridization analysis of recipient cardiac tissues demonstrated that human and murine chromosomes were colocalized in the same cardiomyocytes, indicating that cell fusion occurred between human hematopoietic progeny and mouse cardiomyocytes. These syngeneic- and xenogeneic neonatal transplantations provide compelling evidence that hematopoietic stem/progenitor cells contribute to the postnatal generation of cardiomyocytes through cell fusion, not through transdifferentiation.— Ishikawa, F., Shimazu, H., Shultz, L. D., Fukata, M., Nakamura, R., Lyons, B., Shimoda, K., Shimoda, S., Kanemaru, T., Nakamura, K-i., Ito, H., Kaji, Y., Perry, A. C. F., Harada, M. Purified human hematopoietic stem cells contribute to the generation of cardiomyocytes through cell fusion.


Key Words: NOD/SCID • newborn • plasticity

THE ABILITY OF ADULT STEM CELLS to give rise to cells outside their programmed differentiation pathway (a phenomenon referred to as transdetermination) has attracted much attention because of its therapeutic promise (1 ,2) and because such a high degree of potency was thought to be confined to cells of the early embryo. Although recent reports implicated that bone marrow (BM)-derived cells had the capacity to generate nonhematopoietic cells, it has yet to be determined whether transdetermination is typically an innate, cell-autonomous ability (3 4 5) , or whether it requires fusion with another cell (6 7 8 9 10 11 12) . In cardiac tissue, it has been controversial whether hematopoietic tissue-derived cells can regenerate cardiomyocytes (13 14 15 16 17) , while c-Kit+ cells or Sca-1+ cells reside in cardiac tissue as candidate tissue progenitors (18 19 20) . We therefore set out to investigate the in vivo generation of cardiomyocytes from murine- and human hematopoietic tissues using neonatal transplantation models. The immature environment of neonates may fully potentiate the developmental plasticity of transplanted cells, because the age-related decline of tissue progenitor activity can be restored by the exposure to environmental factors secreted by juvenile hosts (21) . Additionally, to investigate the differentiative capacity of human hematopoietic stem/progenitor cells, we and others reported that neonatal immune-compromised mice supported efficient engraftment of human hemato-lymphoid cells (22 23 24) . In our study, purified murine BM-derived and human cord blood (CB)-derived stem/progenitor cells gave rise to cells that expressed myocyte-specific antigens and showed contractile activity during postnatal development from newborns to adults. Furthermore, the mechanisms underlying mouse and human cardiomyocyte regeneration were clarified by distinguishing donor- and recipient markers with confocal microscopy. These newborn transplantation assays will provide novel tools to examine the developmental plasticity of murine and human stem/progenitor cells in multiple tissues.

MATERIALS AND METHODS

Animals
C57BL/6 mice transgenically expressing enhanced green fluorescence protein (GFP) driven by the cytomegalovirus (CMV) enhancer/chicken ß-actin promoter were kindly provided by Dr. M. Okabe (Osaka University) (25) . C57BL/6 mice transgenically expressing enhanced cyan fluorescence protein (CFP) driven by the CMV enhancer/chicken ß-actin promoter were obtained from the Jackson Laboratory (Bar Harbor, ME). NOD/LtSz-Prkdcscid/PrkdcscidIL2rgnull (NOD/SCID/IL2r{gamma}null) mice were developed by backcrossing complete null mutation at common cytokine receptor {gamma} chain onto NOD-LtSz/scid background (22 ,26) . All the mice were bred and maintained under defined flora. Experiments were performed according to the guidelines established by the Institutional Animal Committee of Kyushu University.

Mouse syngeneic transplantation
Bone marrow cells were harvested from femurs and tibiae of GFP transgenic mice at 8–12 wk of age. Lineage-specific rat antimouse antibodies (B220, CD3{epsilon}, Gr-1, Mac-1, and TER 119, BD Pharmingen, San Jose, CA) and immunomagnetic beads (Dynal, Norway) were used for depleting mature hematopoietic cells. Lin–ScaI+ cells were further purified by cell sorting. 105 Lin–ScaI+ BM cells from GFP mice were injected i.v. into wild-type C57BL/6 mice or C57BL/6 mice transgenically expressing enhanced cyan fluorescence protein (CFP) within 48 h of birth after 400 cGy irradiation. To induce cardiac injury, the cardiac apex of newborn mice was punctured with a 29-gauge needle, followed by the transplantation of donor BM cells.

Analysis of murine BM-derived myocytes
With the recipient mice anesthetized, GFP+ cardiomyocytes were identified in situ with epifluorescence microscopy (Power BX51WI, Olympus, Japan). Kinetic contraction of GFP+ cardiomyocytes was recorded with a high-resolution charge-coupled device (CCD) camera (Cascade 650, Roper Scientific, Trenton, NJ). Following the analysis of contractile efficiency with Metamorph (Roper Scientific, Tucson, AZ), recipients’ cardiac tissue was fixed in 4% paraformaldehyde and sectioned into 50 µm slices with a vibratome or 5-µm slices using a microtome. Each section was examined for the generation of GFP+ myocytes by confocal microscopy (LSM510META, Carl Zeiss, Oberkochen, Germany) before 40 contiguous sections were reconstructed for three-dimensional images. Using the cardiac sections, 240,000~400,000 cardiomyocytes were analyzed for the presence of GFP+ (donor-marker+) cardiomyocytes. To identify the antigenic profile of cardiomyocytes, anti-troponin IC (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-connexin 43 (Chemicon, Temecula, CA) antibodies were used. For transmission electron microscopic analysis, tissue slices were subjected to immunostaining with rabbit anti-GFP antibody (Ab) (Chemicon) for 72 h and with biotin-conjugated anti-rabbit IgG for 48 h. Specimens were then immersed in avidin-biotin complex (ABC) solution (Nacalai tesque, Kyoto, Japan) for 30 min, and visualized by 3,3'-diaminobenzidine (DAB) reaction (Nichirei, Tokyo, Japan). After fixation in 3% glutaraldehyde solution and 2% osmium tetraoxide, the specimens were embedded in epoxy resin (Oken, Tokyo, Japan) and ultrathin sections (0.15 µm) were prepared. In the syngeneic transplantation of GFP+ BM cells into CFP expressing recipients, more than 10 GFP+ cardiomycoytes were tested per recipient to simultaneously analyze the emissions of donor-derived GFP and host-derived CFP by using 458 nm Argon excitation laser. We analyzed 6 CFP Tg recipients transplanted with GFP+ BM cells. Linear unmixing analysis (27) was employed to distinguish GFP+ donor-derived cells, GFP+CFP+ fused cells and CFP+ host-derived cells. Briefly, the emitted fluorescence was detected at 10-nm interval between 450 and 600 nm. The fluorescence intensity at each emitted wavelength was calculated by the equation [S({lambda})SUM = Intensity x S({lambda})CFP + Intensity x S({lambda})GFP] to determine the composition of GFP and CFP.

Xenogeneic transplantation
Human CB mononuclear cells (MNCs) were purchased from Cambrex (Baltimore, MD). Anti-human CD3, CD4, CD8, CD11b, CD19, CD20, CD56, and glycophorin A antibodies were used for the depletion of mature hematopoietic cells. 2–5 x 104 Lin–hCD34+hCD38– cells were i.v. transplanted into newborn NOD/SCID/IL2r{gamma}null mice following 100 cGy irradiation. At ~2–4 mo post-transplantation, hematopoietic chimerism was analyzed by flow cytometric analyses of leukocytes. To distinguish human CB-derived cells from murine cells in cardiac tissues, FISH analyses were performed using Cy3-conjugated human centromeric probe (Cambio, UK) and FITC-conjugated mouse centromeric probe (Cambio). The presence of gap junction was confirmed by the immunostaining for connexin 43. To determine whether the positive signals for centromeres were located inside nuclei and to rule out the possibility of cell overlay (28) , serial X-Y images were obtained from different depths of the specimen at 0.2-µm interval.

RESULTS

Characterization of mouse BM-derived cardiomyocytes
We examined the cardiomyogenic potency of murine BM cells by injecting 106 Lin– BM cells derived from adult C57BL/6 mice transgenically expressing GFP into newborn wild-type C57BL/6 recipients within 48 h after their birth. At ~2–4 mo post-transplantation, GFP+ donor-derived cells accounted for 88.2 ± 5.8% of circulating leukocytes in the recipients. At the time of analysis, recipient cardiac tissue contained GFP+ cells that were of the expected size (30 to 100 µm) for cardiomyocytes of this age and exhibited myocyte-specific striations indistinguishable from those of GFP– cardiomyocytes (Fig. 1 A-C), suggesting the age-dependent maturation of donor-marker+ cardiomyocytes. These striated GFP+ cardiac cells expressed the myocyte-specific markers, troponin I (Fig. 1D-F ) and connexin 43 (Fig. 1G-I ). Contractile function of the GFP+ cells was determined at the single cell concentration by comparing the length at the point of maximal contraction with that of its stretched phase. The contractile efficiency of GFP+ cells was 84.0 ± 2.8% (n=4) compared to values for controls of the lengths of the same cardiomyocytes in stretched phase (Fig. 1J ). Myocyte-specific striations were confirmed in the cytoplasm of the GFP+ cardiac cells by laser-scanning confocal microscopy after the cardiac tissue was fixed with 4% paraformaldehyde (Fig. 1K ). The contractile function of GFP+ cardiomyocytes was further confirmed in vitro after the single-cell suspension of cardiomyocytes was plated in M199 medium (data not shown). The GFP+ cardiomyocytes were further characterized by transmission electron microscopy (TEM) following immunolabeling with anti-GFP Ab. BM-derived cardiomyocytes contained abundant mitochondria (Fig. 1L , arrows), which was essential for sustained contraction. In all cases analyzed, gap junctions bridged GFP+ and GFP– myocytes (Fig. 1L , arrow heads), suggesting that GFP+ myocytes were functionally integrated into the resident network of contractile cardiomyocytes. GFP+ cardiac cells thus possessed the defining morphological, functional, and cytoskeletal characteristics of cardiomyocytes.


Figure 1
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  		Figure 1. Characterization of BM-derived cardiomyocytes in syngeneic transplantation setting. (A, D, G) GFP+ cardiac cells were identified at ~2–4 mo post-transplantation. A–C) Multiple striations are revealed by Normarsky imaging (B shows a merge of A and C). D–F, G–I) Immunolocalization of troponin I (F) and connexin 43 (I) in recipient cardiac sections. J, K) Live GFP cardiomyocyte in contracted (upper) and stretched (lower) phases 0.25 s apart. K) Fixation confirmed the presence of striations in this cardiomyocyte. L) Transmission electron microscopy image of GFP-expressing cardiomyocytes reveals mitochondria (arrows) and gap junctions (arrowheads). M) Distribution of donor-derived cardiomyocytes after Lin– BM cell transplantation following neonatal apical cardiac puncture. Three-dimensional stereotactic images reveal increased numbers of GFP+ cardiomyocytes (red dots) near the site of trauma (arrowhead). Scale bars represent 10 µm.

Because inherent or acquired deficits induce the appearance of hepatocytes (29 , 30) and skeletal myocytes (31 , 32) following BM transplantation, we investigated whether trauma enhanced the appearance of cardiomyocytes in our neonatal transplantation system. 106 Lin– BM cells were i.v. introduced into neonatal mice that had been subjected to apical cardiac trauma (Fig 1M , arrowhead). After preparing the cardiac section of the recipients with or without injury, the number of GFP+ cardiomyocytes was analyzed by laser-scanning confocal microscopy. Injury on cardiac apices of neonatal recipients resulted in an average increase from 9.9 ± 4.3 (n=8) GFP+ cardiomyocytes in noninjured controls to 39.8 ± 16.2 (n=8) GFP+ cardiomyocytes per 40 cardiac sections at 2 mo after injury. This effect was dose-dependent, since transplantation of more (5x106) donor BM cells after trauma resulted in the enhanced appearance of GFP+ cardiomyocytes (79.8±13.3, n=4). The frequency of fused cardiomyocytes detected in the cardiac tissue of the recipients was between 0.01% and 0.04% in accordance with the transplanted cell dose. Three-dimensional images assembled from serial cardiac sections revealed that GFP+ cardiomyocytes were more abundant near the injured apex compared to healthy cardiac sites (Fig. 1M ).

Mechanisms underlying cardiomyocyte regeneration from mouse hematopoietic progenitors
To investigate the mechanisms underlying generation of cardiomyocytes from hematopoietic tissues, we transplanted 105 Lin–ScaI+ mouse BM hematopoietic progenitor cells into recipient mice that constitutively express CFP driven by CMV enhancer/ß-actin promoter. In this transplantation setting, expressions of donor-derived GFP and host-derived CFP on each cardiomyocyte were simultaneously analyzed by laser-scanning confocal microscopy at ~2–4 mo post-transplantation (Fig. 2 A left). Lambda image acquisition detected the signals of both GFP and CFP at different emission wavelengths (450 to 600 nm). Linear unmixing system (Fig 2A , right) determined the composition of GFP and CFP in total fluorescence emitted by each cardiomyocyte based on the equation [S({lambda})SUM= Intensity x S({lambda})CFP + Intensity x S({lambda})GFP] and the reference curves which were obtained from donor GFP+ cells and nontransplanted host CFP+ cells. Using these systems, all the GFP+-striated cardiomyocytes showed fluorescence curve, which was constituted of both GFP and CFP (Fig. 2B ). On the other hand, all the GFP+ hematopoietic cells showed fluorescence curve, which is similar to control GFP+ cells without CFP expressions (Fig. 2B ). The GFP+CFP+ cardiomyocytes were detected irrespective of the exposure to cardiac injury. Thus, in this murine transplantation model, we conclude that the major mechanism underlying the generation of mouse hematopoietic progenitor-derived cardiomyocytes is accounted for by cell fusion.


Figure 2
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  		Figure 2. GFP+ ScaI+ cell-derived cardiomyocytes in CFP+ recipients. A) GFP+ Lin– ScaI+ BM cells were transplanted into neonatal CFP transgenic mice. Lambda image acquisition and linear unmixing analysis were employed to specifically detect donor-derived GFP and host-derived CFP of the samples. Control reference spectra were obtained from cardiomyocytes of host CFP transgenic mice and from BM cells of donor GFP transgenic mice. Total fluorescent light emitted by the samples was collected from 450 nM to 600 nM, and contribution of GFP and CFP was calculated based on the equation [S({lambda})SUM =Intensity xS({lambda})CFP+Intensity xS({lambda})GFP]. If the cell is a product of cell fusion, fluorescence light emitted by the sample is constituted by GFP and CFP. If the cell differentiated from transplanted hematopoietic progenitor cells, the emitted fluorescence light of the cell is equivalent to that of GFP. B) The GFP+ myocytes show the emission spectra, which are composed of both CFP and GFP. The fluorescence intensity in different wavelengths between 450 and 600 nM is shown. The cardiomyocyte in center possesses both CFP and GFP in the cytoplasm. All the GFP+ hematopoietic cells did not express host-derived CFP.

Fusion between human hematopoietic progeny and murine cardiomyocytes in vivo
We further analyzed the in vivo generation of human chromosome+ cardiomyocytes in mouse cardiac tissues. For studying the differentiative capacity of human stem/progenitor cells, we recently developed a novel immune-compromised mouse line by backcrossing complete null mutation of common cytokine receptor (IL2r{gamma}null) onto NOD-scid background (NOD/SCID/IL2r{gamma}null) (26) . This novel scid mouse line exhibits extremely low activity of NK cells as well as complete lack of mature T cells and B cells (26) . The impaired acquired- and innate immunity of NOD/SCID/IL2r{gamma}null mice supported significantly higher levels of engraftment of human cells compared to NOD/SCID/ß2mnull mice (22) . We purified human hematopoietic stem cells (HSCs) from human CB mononuclear cells by cell sorting, and i.v. transplanted ~2–5 x 104 Lin–hCD34+hCD38– cells into neonatal NOD/SCID/IL2r{gamma}null mice (Fig. 3 A, B). At ~2–4 mo post-transplantation, the BM of recipients were highly reconstituted by human HSC-derived cells (Fig. 3C ). The BM of the recipient mice contained human CD34+ stem/progenitor cells (Fig. 3B ) that could reconstitute secondary recipient mice (data not shown). hCD45+hCD34– leukocytes contained multilineage human mature blood cells such as hCD19+ B cells, hCD3+ T cells, and hCD33+ myeloid cells (Fig. 3C ). The majority of circulating leukocytes are also of human origin. Therefore, the hemato-lymphoid systems of the recipient mice were humanized, and adaptive human immunity in recipients may help human CB-derived cardiac cells to survive for long-term in xenogeneic environment. In cardiac tissue of these immunologically humanized mice, we analyzed the generation of human chromosome-containing cardiomyocytes. The human chromosome-containing cardiac cells peripherally expressed connexin 43 and exhibited cardiomyocyte-specific striations (Fig. 3D , left). Serial confocal imaging was used to pinpoint signals for human and murine chromosomes and enable us to exclude the possibility that they arose from cell overlay (28) (Fig. 3D , center and right). For clarifying the origin of each cardiomyocyte in recipient tissue, donor-derived human chromosomes and host-derived murine chromosomes were simultaneously detected on the cardiac specimens by using species-specific chromosome probes in double FISH analysis. In the FISH analyses, we used centromere probes, not sex-chromosome probes, as 5 µm of thin slices may not always include sex chromosomes in the nuclei. Double FISH analysis demonstrated that all human chromosome containing cardiomyocytes possessed murine chromosomes. This result suggests that transplantation of human HSCs resulted in the generation of donor marker+ cardiomyocytes, but that the mechanism underlying generation of HSC-derived cardiomyocyte is due to fusion between donor-derived hematopoietic progeny and host cardiomyocytes, not due to transdifferentiation.


Figure 3
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  		Figure 3. Human HSC-derived cardiomyocytes in NOD/SCID/IL2r{gamma}null recipient mice. A) (Left) NOD/SCID/IL2r{gamma}null recipient mice were used as recipients during their neonatal period. (Right) Facial vein was used as injection route (arrow). B) After the enrichment of hCD34+ cells by immunomagnetic beads, human CB Lin–CD34+CD38– HSCs were purified by cell sorting. C) Flow cytometric analysis was performed to examine the engraftment of human hematopoietic cells in recipient peripheral blood and BM. Recipient BM cells constituted hCD45– mouse blood cells, hCD45+hCD34+ human stem/progenitor cells, and hCD45+hCD34– mature human hematopoietic cells. Human CD19+ B cells, CD3+ T cells, and CD33+ myeloid cells were present in hCD45+hCD34– cell fraction. D) Double FISH analysis was performed on recipient cardiac specimens using human (red) and mouse (green) centromere probes. Nuclei of human- and mouse cells were stained with 4',6'-diam idino-2-phenylidole (DAPI). (Left) Cardiac tissue of the recipient mice was constituted of human chromosome– murine chromosome+ myocytes (upper, labeled as M) and human chromosome+ murine chromosome+ myocytes (upper, marked as F). Cardiomyocytes express connexin 43 peripherally (white dots). (Center and right) Serial confocal images obtained from different depths of the sample confirmed that both human- and murine chromosomes were inside the same nucleus.

DISCUSSION

We have shown that transplantation of purified murine BM- and human CB-derived hematopoietic stem/progenitor cells results in the generation of donor-derived marker+ cardiomyocytes during postnatal development in syngeneic, allogeneic, and xenogeneic environment. Donor-marker+ cardiomyocytes show age-compatible maturation, antigenic profiles and contractile function similar to host-derived myocytes.

The appearance of BM-derived cardiomyocytes was enhanced by cardiac trauma. Trauma-induced regeneration is also exhibited in skeletal myocytes (31) . This suggests that our model recapitulates a general phenomenon in trauma-induced myocyte generation and should allow a dissection of the molecular events regulating postnatal homing, colonization and transdetermination. In fact, further purified BM Lin–ScaI+c-Kit+ HSCs were efficiently mobilized to the injured site of cardiac tissue in the same transplantation setting (data not shown).

The data presented here corroborate the coexistence of conserved (species-independent) classes of a cell fusion-dependent mechanism by which donor-derived marker+ cardiomyocytes can be generated following the transplantation of hematopoietic progenitors. GFP and GFP transgenic mouse have been widely used in biological research (25 , 33) . In the present study, we have used CFP transgenic mice driven by CMV enhancer and ß-actin promoter as recipients. The expressions of CFP and GFP in each cardiomyocyte were simultaneously evaluated by linear unmixing analysis using confocal microscopy, although emission fluorescence spectra of CFP and GFP overlap. This novel approach to use mouse lines harboring distinct fluorescence as donors and recipients and to distinguish donor type- and recipient type-fluorescence by confocal microscopy will provide helpful tools for studying the mechanism of transdetermination in multiple tissues.

In the transplantation of Lin–ScaI+ GFP+ BM cells into newborn CFP recipients, GFP+ cardiomyocytes expressed host-derived CFP in their cytoplasm, indicating the cell fusion-dependent mechanism. Recently, Kajstura et al. reported that murine c-kit+ bone marrow-derived cells gave rise to cardiomyocytes by differentiation, not by cell fusion (34) . The discrepancy may be explained by the differences in transplanted cell type or the nature of injury between their experimental designs and ours. It is also possible that the developmental plasticity of newborn recipients efficiently induces cell fusion between BM-derived or CB-derived hematopoietic progeny and host-derived cardiomyocytes as compared to the adult environment.

The generation of cardiomyocytes from human hematopoietic tissues was examined using the neonatal xenogeneic transplantation models. We and others reported that human hematopoietic cells could engraft efficiently in neonatal immune-deficient mice (23 , 24) . Hemato-lymphoid tissues of a novel immune-compromised mouse line (NOD/SCID/IL2r{gamma}null mouse) could be humanized by the transplantation of purified Lin–hCD34+hCD38– cells (22) . Using these immunologically humanized mice, we showed that the transplantation of purified human CB-derived cells resulted in the generation of human-chromosome+ cardiomyocytes in injured cardiac tissues, which will be helpful for translating stem cell research into clinical regenerative medicine in the future.

Species-specific double FISH analysis revealed that the transplantation of purified human HSCs resulted in the generation of human chromosome+ mouse chromosome+ cardiomyocytes, not that of human chromosome+ mouse chromosome– cardiomyocytes. This result suggests that human HSCs require cell fusion with host cardiomyocytes for contributing to cardiomyocyte generation and that HSCs may not transdifferentiate into cardiomyocytes. The differentiative capacity of human mesenchymal stem cells (MSCs) will be studied in the future using the same transplantation models to determine whether MSCs can give rise to human chromosome+ mouse chromosome– cardiomyocytes via a fusion-independent pathway.

In conclusion, the present data suggested that both human and murine hematopoietic tissues contain the cells that can give rise to cardiomyocytes in vivo. Purified HSCs give rise to cardiomyocytes through cell fusion, not transdifferentiation. The two novel transplantation systems, GFP+ BM donor cells into CFP+ recipient mice and human CB donor cells into T–B–NK– NOD/SCID/IL2r{gamma}null mice, would be powerful tools to study the in vivo capacity of murine- and human stem cells and to gain the insights into transdetermination mechanisms in endodermal or ectodermal lineages (4 , 8 , 10 , 30 , 35 , 36) , as well as clarifying the nature of hematopoietic tissue-derived cardiomyocytes.

ACKNOWLEDGMENTS

This work was supported by grants from Japan Society for Promotion of Science to F.I., the Ministry of Health, Labor, and Welfare of Japan to M.H., Muscular Dystrophy Association and NIH grants (A130389 and HL077642) to L.D.S. The authors thank Mr. Masamichi Ueda and Mr. Hiroshi Fujii for technical assistance.

FOOTNOTES

1 These authors contributed equally to this work.

Received for publication August 12, 2005. Accepted for publication November 2, 2005.

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