Isolation and characterization of SP cells in the heart
Because adult tissue stem cells can be enriched as SP cells (Goodell et al., 1996
; Matsuzaki et al., 2004
), we initially characterized the SP cells in neonatal and adult mouse hearts. FACS analysis demonstrated the presence of cardiac SP cells that are completely blocked by reserpine ( A). SP cells make up 3.5% of heart tissue cells in 2-d-old mice, which is markedly higher than the range of 0.01–1% that was observed in various other organs, including blood, skeletal muscle, and brain (Goodell et al., 1996
). The proportion of heart SP cells decreased rapidly up to postnatal day 7, and made up only 0.02% of heart cells at 6 wk (); this level was consistent with that found in other organs. Cardiac SP cells were characterized further by immunostaining of cytospin preparations with the anti-sarcomeric myosin antibody ( D). 30% of the main population (MP; non-SP) cell fraction contained myosin-positive cardiomyocytes, whereas the SP fraction contained no myosin-positive cells; this provided evidence that cardiac SP cells are nonmyocytes.
Figure 1. Isolation, characterization, and cardiosphere formation of SP cells in the neonate and adult heart. (A) Representative FACS analysis of the 2-d neonatal heart tissues using Hoechst 33342. (B) Postnatal changes in FACS analysis of the SP cell fraction. (more ...)
Cardiac SP cells were phenotyped by analysis of cell surface marker expression. Cell suspensions from P2 mouse hearts were treated with Hoechst 33342 and monoclonal antibodies (Abs). Two-dimensional FACS profiles ( E) revealed that cardiac SP cells are negative for all markers of mature hematopoietic cells, including CD11b, 13, and 45 and Ter119, and are positive for the widely expressing antigens CD29 and CD44. Heterogeneous expression was observed for various immature hematopoietic cell or vascular stem cell markers, such as CD34, c-Kit, Flk-1, and Sca-1. These results provide evidence that the cardiac SP cell fraction is phenotypically immature and is not contaminated by mature hematopoietic cells.
Formation of neurosphere-like spheres by cardiac SP cells in serum-free medium
SP cells were expanded in culture for further characterization. An investigation of various culture procedures led to the successful application of a method that was used previously to generate neurospheres from cultured central nervous system stem cells to generate neurosphere-like spheres from cardiac SP cells. Using this approach, culture of isolated cardiac SP cells resulted in cell division followed by detachment from the culture plate to form a sphere of proliferating cells referred to as a cardiosphere. Cardiospheres were similar in appearance to neurospheres that were derived from cultured central nervous system stem cells, and formed after 7–10 d in serum-free medium in the presence of EGF and FGF2 (, F–I). MP cells also proliferated in serum-free medium, although the population of sphere-initiating MP cells was 100-fold lower than that of cardiac SP cells. Proliferation of cardiospheres was not observed in the absence of EGF and FGF2. Cells within the cardiosphere did not express the cardiac myocyte marker myosin (MF20) or differentiation markers of other mature cell types. To compare the phenotype of cardiospheres with neurospheres, the cells from cardiospheres were immunostained for nestin and Musashi-1, markers of undifferentiated neural precursors. Consistent with neurosphere cells, most cardiosphere cells were positive for nestin and Musashi-1 (, J–N). RT-PCR analysis also confirmed nestin and Musashi-1 mRNA expression in the fetal heart, nonmyocyte fraction, and cardiospheres ( O).
Neonatal and adult cardiac nonmyocytes contain cardiosphere-initiating cells
To investigate the origin of cardiosphere-initiating cells, the nonmyocyte and myocyte-enriched fractions were separated on a Percoll density gradient then assessed in the cardiosphere-forming assay (Reynolds and Weiss, 1992
). Percoll-purified neonatal rat or mouse cardiac nonmyocytes were plated at a density of 10,000–20,000 cells/cm2
on uncoated culture dishes (10 cm in diameter). ~50–100 spheres (1–2 spheres/cm2
) were observed after 7–14 d in vitro. After dissociation and subculture as single cells, ~10% of the primary sphere-derived cells formed secondary spheres. No primary spheres were observed in cardiomyocyte fractions. These results show that cardiosphere-initiating cells are contained within the nonmyocyte cell populations. We also found that cardiosphere formation can be observed in nonmyocytes prepared from adult (10–24-wk-old) murine hearts.
The optimum cell-plating density for formation of cardiospheres was identified as 10,000 cell/ml; most cells that were derived from cardiospheres were positive for nestin and Musashi-1 (unpublished data). This result is consistent with the report by Hulspas et al. (1997)
that spheroid colonies displayed clonal growth when murine neural stem cells were cultured at a density of 10,000 cell/ml. We showed that cardiospheres are derived from a single cell from primary cultures plated at 10,000 cell/ml. Primary cell cultures derived from the neonatal hearts of wild-type mice and mice ubiquitously expressing GFP (Okabe et al., 1997
) were mixed at a ratio of 9:1. Resultant cardiospheres were GFP-negative or GFP-positive; there was no evidence of mixing of the GFP-positive and GFP-negative cells (unpublished data).
Differentiation of cardiosphere-derived cells into multiple cell types in vitro
The multipotent capacity of cardiosphere-derived cells was investigated by dissociating cardiospheres to form a single-cell suspension, and then assessing the ability of these cells to differentiate in the absence of EGF and FGF2. Because cardiospheres are similar to neurospheres, the capacity of cardiosphere-derived cells to differentiate into neurons, glial cells, and cardiomyocytes was investigated. At day 0, most cardiosphere-derived cells were positive for nestin and Musashi-1 (). At this stage, most cells were small in size, and did not express microtubule-associated protein 2 (MAP2), glial fibrillary acidic protein (GFAP), or myosin heavy chain (MHC) (). By day 14, the cells had lost the ability to express nestin and Musashi-1 (). Differentiation of the cardiosphere-derived cells was associated with the induction of various morphologic changes, including the formation of neuron-like dendrites and the initiation of spontaneous beating within several weeks, a characteristic feature of cardiomyocytes. Cells within the differentiated population stained positively with anti-MHC, anti-GFAP, or anti-MAP2 Abs. The expression of markers for stem/progenitor cells, including nestin, Musashi-1,
(multi-drug resistance transporter gene 1
) (Zhou et al., 2001
), and for differentiated cells, including MAP2
, and β-MHC,
was assessed by RT-PCR ( L). Stem/progenitor cell marker genes were expressed at day 0, but their expression gradually decreased and could not be detected at day 14. By comparison, expression of GFAP
, and β-MHC
were observed from day 7, which provided evidence of the differentiation of cardiosphere-derived cells into neurons, glia, and cardiomyocytes.
Figure 2. Differentiation of cardiosphere-derived cells in vitro. Cardiospheres were obtained from P2 neonates. Dissociated cardiosphere-derived cells were maintained in medium without EGF and FGF-2. At day 0, almost all cells stained with antinestin (A) and anti–Musashi-1 (more ...)
Cardiosphere differentiation into peripheral nerve cells
The ability of cardiospheres to differentiate into cells with PNS characteristics also was examined. Differentiated cardiospheres were immunostained with peripherin, a PNS neuronal marker; p75, a common receptor subunit of the nerve growth factor family that is expressed in sensory neurons, neural crest stem cells (Morrison et al., 1999
), and Schwann cells (Stemple and Anderson, 1992
); and MAP2, a pan-neuronal marker. A population of small cardiosphere-derived cells with long axons stained positive with all antibody markers (, A–C). Immunostaining with anti-Hu (a pan-neuronal marker) showed strong expression of Hu in particular small cells, which confirmed their differentiation into a neuronal cell type ( D).
Figure 3. Analysis of the expression of neuron-specific markers in cardiosphere-derived cells. Cardiospheres obtained from P2 neonates were induced to differentiate as described in “Materials and methods.” (A–D) Immunostaining of the neural (more ...)
α-Tubulin promoter (Tα-1)–EYFP transgenic mice, in which all cells with a neuronal lineage express enhanced yellow fluorescent protein (EYFP), were used to evaluate differentiation of cardiosphere-derived cells into neuronal cell types (Sawamoto et al., 2001
). Differentiated cardiospheres from Tα-1-EYFP transgenic mice were immunostained with the anti-MAP2 antibody, and revealed expression of MAP2 by the EYFP+
cardiosphere-derived cells ().
RT-PCR of MASH1
, a proneuronal basic helix-loop-helix protein expressed in immature neuronal cells (Ross et al., 2003
; and P0
, a Schwann cell myelin marker (Lemke and Axel, 1985
; Lemke et al., 1988
), also confirmed the differentiation of cardiosphere-derived cells into neuronal cell types ( G). Differentiation of cardiosphere-derived cells leads to a reduction in p75
expression, and a reduction—followed by a loss—of MASH1
expression. By comparison, expression of P0 was induced as a result of cardiosphere cell differentiation. These findings are consistent with the differentiation phenotype of neuronal cells of the PNS lineage.
Cardiosphere differentiation into cardiomyocytes and smooth muscle cells
Cardiospheres and cells dissociated from cardiospheres express GATA4, but not Nkx2.5 or muscle enhancement factor 2C, which indicate that these cells are not cardiomyocytes, but their early progenitors ( A). The cardiac-specific genes, ANP, Cav1.2, or α-skeletal actin, are activated in cardiosphere-derived cells 7 d after dissociation, whereas spontaneously beating cells are evident at 14 d. B shows the representative action potentials recorded from the cardiosphere-derived cardiomyocytes at day 14. Compared with mature cardiomyocytes, the resting potential was shallower but the duration of the action potential was similar. Immunofluorescent staining for Nkx2.5, GATA4, and actinin was evident in particular cells at day 14 (, C–H).
Figure 4. Characterization of cardiosphere-derived cells as stem cells for cardiomyocytes and smooth muscle cells. Cardiospheres obtained from P2 neonates were induced to differentiate as described in “Materials and methods.” (A) RT-PCR analysis (more ...)
In the present study, the capacity of cardiosphere-derived cells to differentiate into smooth muscle was investigated by examining the production of α-smooth muscle actin (α-SMA) and calponin (, J–M). At day 0, α-SMA+
cells represented 11.8 ± 4.8% of total cells and were negative for calponin. By day 14, the population of α-SMA+
cells had increased to 42.9 ± 16.2% (Table SI; available at http://www.jcb.org/cgi/content/full/jcb.200504061/DC1
); most of these were positive for calponin. These findings show that the cardiosphere-derived cell population consists largely of stem/progenitor cells; a small fraction has the capacity to differentiate, at least in part, into cardiomyocytes and smooth muscle cells.
Cardiosphere cells behave like neural crest cells in vivo
At day 0, 99.8% of the cardiosphere-derived cells were nestin+
, a characteristic of stem/progenitor cells (Table SI). Following differentiation, the expression of these markers is lost and is replaced by the expression of various markers for differentiation. Many cells were positive for GFAP (68.1 ± 1.8%) and α-SMA (42.9 ± 16.2%), whereas the population of neurons and cardiomyocytes represented 0.45 ± 0.21% and 0.28 ± 0.17% of the total cell population, respectively. Differentiated neurons and cardiomyocytes did not express nestin. The capacity of cardiosphere-derived cells to generate neurons, glia, and smooth muscle cells in culture suggests that they have neural crest-like characteristics. To investigate these characteristics further, we tested the behavior of cardiosphere-derived cells in vivo. Because neural crest cells that originate from the 1–3 somite level contribute to heart structures in the chick embryo, one to three DiI (a lineage tracing dye)-labeled cardiospheres were transplanted into the migration staging area (MSA) between the dorsal neural tube and somite at the first and/or second somite level of Hamburger and Hamilton (1951)
stage 9 chicken embryos. DiI-labeled cardiosphere-derived cells were well-dispersed in the embryonic environment 24 h after transplantation ( A). Many cardiosphere-derived cells seemed to migrate along with host-derived human natrual killer-1 (HNK1)–positive neural crest cells (). Rodent neural crest cells do not express HNK1. Because DiI is located at the cell surface membrane, the border zone area between the DiI-labeled cardiosphere-derived cells and host-derived HNK1-positive cells appears yellow when many donor cells are concentrated among the recipient cells. Medially migrating cardiosphere-derived cells were found in the developing PNS, such as the dorsal root ganglion ( B) and the ventral spinal nerve ( D). Cardiosphere-derived cells also entered the lateral migration pathway that normally is taken by neural crest-derived melanocyte precursors, ectomesenchymal cells, and those migrating into the cardiac region. Very few cells reached the heart region.
Figure 5. Cardiosphere cells behave as neural crest in the chicken embryonic environment. Cardiospheres obtained from P2 neonates were labeled with DiI, and transplanted into the chick neural crest. (A) A dorsal view of a chicken embryo that received cardiosphere (more ...)
To facilitate the migration of cardiosphere-derived cells into the heart region, DiI-labeled cardiospheres were transplanted directly onto the lateral pathway between the dorsal somite and the overlying epidermal ectoderm. Again, cardiosphere-derived cells were well-dispersed in the embryonic environment 24 h after transplantation ( E). 48 h later, the labeled cells successfully entered the out-flow tract and the conotruncus of the developing heart ( F). On transverse sections of transplanted embryos, cardiosphere-derived cells were found in the heart region along with HNK1-positive host-derived cardiac crest cells (′). To examine further the migratory capacity of cardiosphere-derived cells in vivo, labeled cardiospheres were transplanted into the MSA at the wing level of stage 12–13 embryos. 48 h after transplantation, cardiosphere-derived cells seemed to migrate along the medial pathway ( H). On transverse sections, many cardiosphere-derived cells were found to contribute to the PNS, such as the sympathetic ganglia (′), and also to dorsal root ganglia and spinal nerves (not depicted). These results show that cardiosphere-derived cells behave like neural crest cells and migrate in the chick embryonic environment.
To examine the differentiation of transplanted cardiosphere-derived cells in vivo, cardiospheres were prepared from GFP-expressing rat SP cells, because the GFP label allows longer-term identification of individual cells. GFP-labeled cardiospheres were transplanted as described above, and the distribution and differentiation of cardiosphere-derived cells were assessed 2 d later. Many cardiosphere-derived cells formed peripheral ganglia and many also expressed the neuronal marker Hu (, A–G). Expression of the glial marker GFAP at the trunk and cranial levels also was evident (, H–K). Several cardiosphere-derived cells also contributed to the major blood vessels and expressed smooth muscle actin (, L–O). These results indicate that cardiosphere-derived cells have the capacity to contribute to neural crest-derived tissues in the chick embryo.
Figure 6. Differentiation of GFP-labeled cardiosphere-derived cells in the chick embryonic environment. Cardiospheres obtained from P2 neonates of the GFP-Tg mice were transplanted into chick neural crest. Cardiospheres were transplanted into the MSA at trunk level (more ...)
Distribution and differentiation of neural crest-derived cells in the heart
For the analysis of the neural crest cell lineage, Yamauchi et al. (1999)
generated transgenic mice harboring a Cre gene driven by a promoter of P0, and crossed P0-Cre transgenic mice with CAG-CAT-Z indicator transgenic mice, which carry a lacZ gene downstream of a chicken-actin promoter and a “stuffer” fragment flanked by two loxP
sequences. In three different P0-Cre lines crossed with CAG-CAT-Z transgenic (Tg), embryos carrying both transgenes showed lacZ
expression in tissues that were derived from neural crest cells, such as spinal dorsal root ganglia, sympathetic nervous system, enteric nervous system, and ventral craniofacial mesenchyme at stages later in development than E9.0.
Double Tg mice carrying P0-Cre recombinase and CAG-CAT-EGFP transgenes showed EGFP expression in tissues that were derived from neural crest cells, including spinal dorsal root ganglia, sympathetic nervous system, enteric nervous system, and ventral craniofacial mesenchyme (unpublished data). EGFP+ cells were concentrated at the outflow tract between the aortic and pulmonary arteries and the aortic valves, and at the intramuscular and subepicardial layer of both ventricles, including the intraventricular septum, free wall, and apex (, A–E). EGFP+ cells also were observed at the atrial wall. Triple immunostaining of the heart at E17.5 showed a lack of staining with the anti-actinin antibody (, F–H) and evidence of staining with the anti-GATA4 antibody ( I). Some EGFP+ cells stained with anti-nestin antibody (). Triple immunostaining of the heart of 10-wk-old mice clearly demonstrated that a portion of the GFP+ cell population were actinin+ cardiomyocytes (). Consistent with these results, the cardiosphere-derived cells from 10-wk-old double transgenic mice hearts were clearly GFP+ ( N). These findings strongly suggest that neural crest-derived cells remain in the heart as stem cells in adults and have the capacity to differentiate into various cell types, including cardiomyocytes.
Figure 7. Distribution and coimmunostaining of neural crest derived cells in the heart. (A–E) P0 Cre/CAG-CAT-EGFP double Tg mice heart was immunostained with Toto-3 and anti-GFP antibody. Distribution of EGFP+ cells in the heart was demonstrated. EGFP+ (more ...)