HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-4863fjev1 20/7/950    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ishikawa, F. Articles by Harada, M. Search for Related Content PubMed PubMed Citation Articles by Ishikawa, F. Articles by Harada, M.

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, Bonnie Lyons^||, Kazuya Shimoda^*, Shinji Shimoda^*, Takaaki Kanemaru^*, Kei-ichiro Nakamura, 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;

Second Department of Anatomy, Kurume University School of Medicine, Kurume, Japan;

Zeiss Japan, Tokyo, Japan;

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

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

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

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

SPECIFIC AIMS

We aimed to analyze the cardiomyogenic potential of murine and human hematopoietic stem/progenitor cells and to determine the mechanism underlying the cardiomyogenesis from hematopoietic tissues. For this purpose, we developed novel transplantations strategies: (1) transplantation of enriched GFP⁺ murine bone marrow progenitor cells into cyan fluorescence protein (CFP)-expressing mice and (2) transplantation of Lin^–CD34⁺CD38^– human cord blood hematopoietic stem cells into a T^–B^–NK^– immune-deficient NOD-scid/IL2r^null mouse.

PRINCIPAL FINDINGS

1. Mechanisms underlying cardiomyocyte regeneration from mouse hematopoietic tissues
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 cytomegalovirus enhancer/ßbeta;-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. 1 A left). Lambda image acquisition detected the signals of both GFP and CFP at different emission wavelengths (450 nM to 600 nM). Linear unmixing system (Fig 1A , right) determined the composition of GFP and CFP in total fluorescence emitted by each cardiomyocyte based on the equation [S()_SUM=IntensityxS()_CFP+IntensityxS()_GFP] and the reference curves, which were obtained from donor-derived GFP⁺ cells and host-derived CFP⁺ cells. Using these systems, all the GFP⁺-striated cardiomyocytes showed fluorescence curve, which was constituted of both GFP and CFP (Fig. 1B ). The GFP⁺CFP⁺ cardiomyocytes were detected irrespective of the exposure to cardiac injury. On the other hand, all the GFP⁺ hematopoietic cells showed fluorescence curve, which is similar to control GFP⁺ cells without CFP expressions (Fig. 1B ). Thus, in the murine syngeneic transplantation model, we conclude that the major mechanism underlying the generation of mouse hematopoietic progenitor-derived cardiomyocytes is accounted for by cell fusion.

View larger version (56K): [in this window] [in a new window]   Figure 1. 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()SUM =Intensity xS()CFP+IntensityxS()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.

2. Fusion between human hematopoietic progeny and murine cardiomyocytes in vivo
We further analyzed the in vivo generation of human 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 (IL2rg^null) onto NOD-scid background (NOD/SCID/IL2r^null). We i.v. transplanted 2–5 x 10⁴ human CB-derived Lin^–hCD34⁺hCD38^– HSCs into neonatal NOD/SCID/IL2r^null mice. At 2–4 mo post-transplantation, the BM of recipients were highly reconstituted by human cells (data not shown). The BM of the recipient mice contained human CD34⁺ stem/progenitor cells (data not shown) 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 (data not shown). 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⁺ cardiac cells peripherally expressed connexin 43 and exhibited cardiomyocyte-specific striations (Fig. 2 , 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 (Fig. 2 , 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 fluorescence in situ hybridization (FISH) analysis. 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 maker⁺ cardiomyocytes, but that the mechanism is due to fusion between donor-derived hematopoietic progeny and host cardiomyocytes, not due to transdifferentiation.

View larger version (28K): [in this window] [in a new window]   Figure 2. Human HSC-derived cardiomyocytes in NOD/SCID/IL2rnull recipient mice. 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.

CONCLUSIONS AND SIGNIFICANCE

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 and xenogeneic environment.

The data presented here corroborate the coexistence of conserved (species-independent) classes of cell-fusion-dependent mechanism by which donor-derived marker⁺ cardiomyocytes can be generated following the transplantation of hematopoietic progenitors. The expressions of CFP and GFP in each cardiomyocyte were simultaneously evaluated by linear unmixing analysis using confocal microscopy. This novel approach to use mouse lines harboring distinct fluorescence as donors and recipients (Fig. 3 A) 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.

View larger version (30K): [in this window] [in a new window]   Figure 3. Schematic Diagram. A) In the syngeneic transplantation setting, BM Scal+ cells derived from GFP transgenic mice were injected into CFP transgenic recipients. The cells generated by differentiation express GFP, but no CFP. The cells generated by fusion express both GFP and CFP. B) In the Xenogeneic transplantation setting, human CB HSCs were injected into NOD/SCID/IL2rgnull mice. The cells generated by differentiation possess human chromosomes, but not murine chromosomes. The cells generated by fusion possess both human and murine chromosomes.

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

Transplantation of purified human HSCs resulted in the generation of human chromosome⁺ mouse chromosome⁺ cardiomyocytes, not that of human chromosome⁺ mouse chromosome^– cardiomyocytes (Fig. 3B ). 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.

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 KO 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 other tissues, as well as clarifying the nature of hematopoietic tissue-derived cardiomyocytes.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4863fje

This article has been cited by other articles:

				A. Orlandi, F. Pagani, D. Avitabile, G. Bonanno, G. Scambia, E. Vigna, F. Grassi, F. Eusebi, S. Fucile, M. Pesce, et al. Functional properties of cells obtained from human cord blood CD34+ stem cells and mouse cardiac myocytes in coculture Am J Physiol Heart Circ Physiol, April 1, 2008; 294(4): H1541 - H1549. [Abstract] [Full Text] [PDF]

				E. Miyata, M. Masuya, S. Yoshida, S. Nakamura, K. Kato, Y. Sugimoto, T. Shibasaki, K. Yamamura, K. Ohishi, K. Nishii, et al. Hematopoietic origin of hepatic stellate cells in the adult liver Blood, February 15, 2008; 111(4): 2427 - 2435. [Abstract] [Full Text] [PDF]

				A. Dedja, T. Zaglia, L. Dall'Olmo, T. Chioato, G. Thiene, L. Fabris, E. Ancona, S. Schiaffino, S. Ausoni, and E. Cozzi Hybrid cardiomyocytes derived by cell fusion in heterotopic cardiac xenografts FASEB J, December 1, 2006; 20(14): 2534 - 2536. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-4863fjev1 20/7/950    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ishikawa, F. Articles by Harada, M. Search for Related Content PubMed PubMed Citation Articles by Ishikawa, F. Articles by Harada, M.