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Circulation Research. 2007;100:e1-e11
Published online before print November 22, 2006, doi: 10.1161/01.RES.0000253487.02398.85
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(Circulation Research. 2007;100:e1.)
© 2007 American Heart Association, Inc.

UltraRapid Communication

Bone Marrow Oct3/4+ Cells Differentiate Into Cardiac Myocytes via Age-Dependent Paracrine Mechanisms

Benedetta A. Pallante, Inga Duignan, Daniel Okin, Andrew Chin, Michael C. Bressan, Takashi Mikawa, Jay M. Edelberg

From the Departments of Medicine (B.A.P., I.D., D.O., A.C., M.C.B., J.M.E.) and Cell and Developmental Biology (J.M.E.), Weill Medical College of Cornell University, New York; and Cardiovascular Research Institute and Department of Anatomy (T.M.), School of Medicine, University of California, San Francisco.

Correspondence to Jay M. Edelberg, GlaxoSmithKline. E-mail jay.m.edelberg{at}gsk.com


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The mechanisms that govern the capacity of the bone marrow stem cells to generate cardiac myocytes are still unknown. Herein we demonstrate that the cardiomyogenic potential of bone marrow–derived Oct3/4+/cKit+/–/CXCR4+/–/CD34–/Sca1– cells is governed by age-dependent paracrine/juxtacrine platelet-derived growth factor (PDGF) pathways. Specifically, bone marrow cell cultures from both 3- and 18-month-old mice formed aggregates of Oct3/4+ cells circumscribed by PDGFR{alpha}+/Oct3/4–/Sca1+ cells. In young (3-month) bone marrow cell cultures, induction of PDGF-AB preceded the induction of cardiac genes and was required for the generation of cardiomyogenesis. Indeed, in old (18-month) cultures, diminished PDGF-B induction was associated with impaired cardiomyogenic potential, despite having Oct3/4 levels similar to those in the young cells. Importantly, supplementation with PDGF-AB specifically restored the cardiac differentiation capacity of the old bone marrow cells. Together these results demonstrate that, regardless of age, the bone marrow niche contains Oct3/4 stem cells that are capable of differentiating into cardiac myocytes. Moreover, this differentiation is governed by age-dependent PDGF-AB–mediated paracrine/juxtacrine pathways that may be essential in the translation of bone marrow cell–mediated cardiomyogenesis.


Key Words: bone marrow • stem cells • myogenesis • Oct3/4 • PDGF


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Previous studies have shown that bone marrow cells may have the ability to give rise to cells of the heart, including cardiac myocytes.1–3 Experimentally this process is notable, however, for the low survival rates4,5 and the limited differentiation efficiency2,3 of the bone marrow cells in the cardiac tissue. Moreover, evidence of the fusion of bone marrow cells to preexisting cardiac myocytes in the heart has called into question the potential of bone marrow cells to generate new cardiac myocytes.6,7 Thus, to address this controversy, we elected to use an in vitro approach to define the cell population(s), factors, and mechanisms by which adult bone marrow cells may give rise to cardiac myocytes.

Studies demonstrating that the adult bone marrow contains highly plastic cells with multilineage differentiation potential8,9 have suggested that bone marrow may be capable of giving rise to myocyte-like cells.10–12 The differentiation of these bone marrow cells into cardiac myocytes may be highly dependent on microenvironmental and trophic signals. Specifically, recent reports have highlighted the importance of inductive extracellular cues in the cardiac differentiation of adult stem cells.13,14 In particular, endothelial progenitor cells (EPCs) and EPC-derived growth factors have been shown to enhance cardiac tissue regeneration mediated by resident cardiac stem cells.13,15 Moreover, endothelial cells have been shown to direct embryonic stem (ES) cell cardiac differentiation,16 suggesting that endothelial cell/EPC trophism may regulate bone marrow–mediated cardiomyogenesis.

Previous work performed in our group has focused on the role of platelet-derived growth factor (PDGF) as an important mediator of the cardiac myocyte-endothelial/EPC interactions that govern cardiac vascular homeostasis and ischemic response in the adult heart.17,18 Indeed, the age-related impairment in PDGF-B induction pathways underlies a senescent predisposition to increased cardiac injury but can be reversed through targeting the functions of PDGF in the heart.18 Specifically, PDGF-AB is cardioprotective, acting as both as a proangiogenic and antiapoptotic agent. Moreover, we have found that PDGF-AB promotes the formation of bone marrow cell–derived myocardial bundles in rat models of myocardial infarction.10 These data together with the ability of PDGF to increase cardiac differentiation of murine ES cells19 suggest that PDGF pathways may be important in the cardiac differentiation of the highly plastic cells of the adult bone marrow.

Herein we describe the use of an in vitro system to identify the source of cardiomyogenic potential in the bone marrow and dissect the mechanisms regulating bone marrow cell cardiac differentiation. Importantly, the ability of bone marrow cell cultures to give rise to cardiac myocytes was confirmed by their spontaneous chronotropic activity, the expression of a range of cardiac genes and their electrophysiological properties. We then demonstrated that bone marrow–derived cardiac myocytes originate from aggregates of Oct3/4+/cKit+/–/CXCR4+/–Sca1–/CD34– cells in culture. Furthermore, we found that the cardiac myocyte differentiation of these aggregates is related to the age-associated changes in PDGF pathways. Specifically, cultures of bone marrow cells from both young (3-month) and old (18-month) mice gave rise to Oct3/4+ aggregates surrounded by PDGF receptor (R){alpha}+ cells. In the young cultures, PDGF-B expression preceded the induction of cardiac myocyte differentiation. In the old cell cultures, a marked reduction in the PDGF-B induction was associated with a significant decrease in cardiac myocyte gene expression. Importantly, addition of PDGF restored the cardiomyogenic induction of the aging cultures, demonstrating the importance of local microenvironmental cues in the differentiation of bone marrow derived cells into functional cardiac myocytes.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
An expanded Materials and Methods section is included in the online data supplement.

Statistical Analysis
Results are presented as mean±SEM. Mean group values were compared by using unpaired and paired t tests. A repeated-measures ANOVA with post hoc testing was also performed (SPSS software) (Figure 4C). Probability values of P<0.05 were considered statistically significant. A detailed description is included in the online data supplement.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Bone Marrow Cells Differentiate Into Functionally Active Clusters of Cardiac Myocytes
Culturing bone marrow cells under conditions previously reported to promote bone marrow derivation of chronotropic activity and expression of {alpha} (adult)- and ß (fetal)-myosin heavy chain (MHC),10 confirmed the differentiation of the bone marrow cells into clusters of cardiac myocytes (CCMs) as demonstrated using a panel of molecular and protein makers. Specifically, immunocytological (Figure 1A) and RT-PCR analysis (Figure 1B) of both bone marrow cell cultures and isolated spontaneously contracting cells revealed the expression of the following cardiac markers: the sarcomeric proteins cardiac troponin-T (cTnT), {alpha}-sarcomeric actinin, {alpha}-cardiac actin, {alpha}-MHC, ß-MHC, light chain ventricular myosin (MLC-2V); the transcription factor Nkx2.5/csx; the gap junction proteins connexin43 (Cx43) and connexin40 (Cx40). The cardiac myocyte differentiation of the bone marrow cells was associated with the expression of ß1- and ß2-adrenergic receptors, which are critical in the regulation of cardiac chronotropic function (Figure 1C and 1D).20 Indeed, this cardiac gene profile correlated with observational studies of spontaneous chronotropy (Videos 1 and 2 in the online data supplement) in the newly formed CCMs (60.1±12.2 contractions per minute).


Figure 1
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  		Figure 1. Bone marrow cells differentiate into functionally active cardiac myocytes. A, Immunostaining of day-14 spontaneously beating clusters of cardiac myocytes (CCMs) with anti-cTnT (Texas red), anti-Cx40 (fluorescein isothiocyanate, green), and anti–{alpha}-sarcomeric actinin (Texas red). Fluorescent images also show the DNA dye 4',6-diamidino-2-phenylindole (DAPI) (blue). Bars=20 µm. B, RT-PCR analysis of day-0 to -14 whole bone marrow cells (BM) cultured with FGF-b and VEGF and of day-14 CCMs showing expression of the cardiac genes Nkx2.5/csx, {alpha}-cardiac actin, and fetal/ß- and adult/{alpha}-MHC, light chain ventricular myosin (MLC-2V), Cx43, and Cx40. Controls: heart (positive); no cDNA (negative). C and D, Immunostaining of day-14 CCMs (C) and RT-PCR analysis of day-0 to -14 whole bone marrow cell cultures (BM) showing the expression of the ß1- and the ß2-adrenergic receptors (AR) (D). Fluorescent images also show the DNA dye DAPI (blue). Bar=20 µm. E, Reactivity to adrenergic stimulation was assessed by placing day-7 CCMs (arrow) into a microelectrode array chamber with 60 electrodes (arrowhead) (a) and recording the electric tracing (b) before (c) and after (d) stimulation with 1 µmol/L isoproterenol-HCl.

Based on the development of the bone marrow cells into phenotypic cardiac myocytes, day-7 CCMs were selected for functional analysis. Data were acquired using a microelectrode array (Figure 1E, a and b), before (Figure 1E, c) and after (Figure 1E, d) stimulation with 1 µmol/L isoproterenol, showing an increase in the depolarization rate, depolarization amplitude, and maximum electropotential amplitude (Table 1). Together, these findings confirmed the reactivity of CCMs to ß-adrenergic stimulation and thus the phenotypic function of the bone marrow–derived cardiac myocytes.


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  		Table 1. Microelectrode Array Results Showing Effects of ß-Adrenergic Stimulation With Isoproterenol on Bone Marrow–Derived Cardiac Myocytes

Cardiac Myocytes Derive From Oct3/4+ Cells, Forming Stem Cell Aggregates or Bone Marrow–Derived Spheres
Observational studies revealed that clusters of cTnT+ cells (28.6±4.5% of total CCMs cells) form at day 7 at the periphery of spherical cellular aggregates, termed bone marrow spheres (BMSs) (Figure 2A, c and d). The number of these BMSs reached a peak at day 7 (6.0±1.9 BMS/animal, day 5; 10.7±1.9, day 7; 5.8±2.3, day 14), when spontaneous chronotropic activity was also observed in {approx}1/3 of the BMSs (32.9±10.6%). The size of the CCMs budding from the periphery of the BMSs gradually increased between days 10 and 14, when they detached and were observed in suspension (Figure 2A, e and f).


Figure 2
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  		Figure 2. Cardiac myocytes derive from Oct3/4+ stem cells organized in BMSs. A, Whole bone marrow cell cultures (a), negative for cTnT on day-3 after plating (Texas red [TR]) (b), formed BMSs between days 5 and -7 (arrows) (c and d). Clusters of spontaneously beating cardiac myocytes (CCMs), positive for cTnT (TR) (c, inset, and f), originated at the periphery of day-7 BMSs (c and d, arrowheads) and were released in suspension by day 14 (e and f). Bar=50 µm. B, Purified day-5 BMSs, cultured on fibronectin, gave rise at day 21 to cTnT+ CCMs (a and b, arrowheads; TR). c and d, CCMs generated by mixing ß-Gal+ cells, deriving from day-5 BMSs purified from ROSA-26 bone marrow cell cultures and day-5 whole bone marrow cell cultures, derived from wild-type/ß-Gal– mice. Cultures were stained at day 14 for ß-Gal (blue) (c), cTnT (TR) (c and d), and the DNA dye DAPI (blue) (d). Colocalization of ß-Gal+/cTnT+ in day-14 CCMs (arrowheads) confirmed that cardiac myocytes derive from BMSs. Bars=50 µm. C, RT-PCR and quantitative RT-PCR analysis performed on purified BMSs confirmed expression of Oct3/4 and other stem cells genes (positive controls: ES cells, ovary; negative control: liver). Quantitative RT-PCR data are expressed as arbitrary units (AU), 1 AU=day-7 BMS expression levels. Bars represent mean AU±SEM (3 samples/group; 20 to 30 BMS/sample). D, Day-7 and day-14 BMSs (BMSd7, BMSd14) were stained for Oct3/4 (fluorescein isothiocyanate [FITC]) (e and f) and nestin (FITC) (g). Positive controls for Oct3/4: oocyte (c) and ES cells (d). Negative controls for Oct3/4: cumulus cells (b) counterstained with DAPI (a). Isotype controls: for Oct3/4 (f, inset) and nestin (g, inset). Bar=50 µm. E, Confocal images showing 3 overlaying sections (a, b, and c) of a day-14 BMS stained for cTnT (Cy3; red, cytoplasm), Oct-3/4 (FITC; green, nucleus), and the DNA dye To-pro-3 (blue). Images show that cTnT+ cells (arrows), localized at the periphery of the BMS, radiate from Oct3/4+ cells (arrowheads), localized in the center of the BMS. d through f, Detail of differentiating cardiac myocytes confirming that the expression of Oct3/4 (arrowheads) is gradually followed by expression of cTnT (arrows). Coexpression of low levels of Oct3/4 and cTnT was occasionally observed in differentiating cells. F, PCR and quantitative RT-PCR results showing that reduction of Oct3/4 expression, 24 hours after (day-3) targeted siRNA in bone marrow cell cultures, reduced the expression of the cardiac marker ß-MHC (day-7). Controls: cultures treated with scrambled siRNA duplexes. Paired t test (N=6); **P≤0.02, ***P≤0.005. Data are expressed as mean relative values (RV)±SEM.

Notably, the formation of BMSs and subsequent CCMs was highly dependent the concentration of cells at the time of initial plating. Specifically, serial dilution studies revealed that cell density is critical in the formation of BMSs and CCMs chronotropy (Table 2). The highest number of BMSs was observed in the wells with 7.0x106 cells/well, whereas at lower concentrations (3.5x106 to 1.7x106 cells/well) few BMSs were formed and none developed chronotropic activity.


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  		Table 2. Effect of Cell Density on Day-7 BMS Numbers and Chronotropy

Experiments were then conducted to determine the mechanisms governing the generation of cardiac myocytes from the BMSs. To isolate the BMS cells from the non-BMS cells, spheres were selected by microdissection at day 5, before the onset of chronotropic activity and cultured on fibronectin. Onset of chronotropic activity was observed at day 14 (Figure 2B, a and b) in {approx}1/3 of the BMSs (N=7/24). In addition, approximately one-third of the cell clusters in suspension at day 21 were found to be cTnT+ (N=67/214). These findings represent a 1-week delay in the onset of chronotropic activity and CCMs formation, compared with control cultures, suggesting that an interaction with cells and/or extracellular signals outside of the BMS may be important for cardiac myocyte differentiation. Alternatively, we considered that the mechanical disruption of the BMS might account for the delay in cardiac myocytes generation. To test this, genetically labeled LacZ BMSs were isolated at day-5 whole bone marrow cell cultures prepared from ROSA-26 mice, dissociated into single-cell suspensions and mixed with intact wild-type whole bone marrow cell cultures forming BMSs. Notably, the presence of cTnT+/ß-galactose+ (ß-Gal+) cells in day-14 CCMs, which were composed predominately of ß-Gal– cells, and the lack of cTnT+/ß-Gal+ cells alone (Figure 2B, c and d), supported the importance of microenvironmental interactions in the generation of bone marrow–derived cardiac myocytes.

The temporal patterns of cardiac differentiation and the morphology of the BMSs were similar to those observed in studies of ES cell–derived embryoid bodies16 and cardiospheres derived from resident cardiac stem cells.21,22 Based on these parallels, molecular and immunocytochemical studies were conducted to assess the stem cell nature of the BMSs capable of giving rise to the cardiac myocytes. RT-PCR analysis of the bone marrow–derived BMSs demonstrated the expression of Oct3/4 as well as the stem cell markers nanog, Dppa3 (developmental pluripotency-associated 3), and prominin-1/AC133 and the progenitor cell marker nestin (Figure 2C). Quantitative RT-PCR revealed that the levels of Oct3/4, nanog, and Dppa3 in the day-7 BMSs were {approx}10% of those observed in ES cells. Between day 7 and 14, there was a significant down regulation in the expression of these genes (unpaired t test; Oct3/4, P=0.04; nanog, P=0.019; Dppa3, P=0.005) as well as prominin-1 (P=0.04). Notably, however, immunostaining revealed the continued presences of Oct3/4 in the day-7 and -14 BMSs (Figure 2D, e and f). Conversely, the levels of expression of nestin increased in day-14 BMSs compared with day 7 (P=0.003), indicating a possible transition from a more undifferentiated state to a progenitor state committed to the cardiac lineage (Figure 2D, g).

Derivation of cardiac myocytes from Oct3/4+ cells localized in the BMSs was then confirmed by confocal imaging of day-14 BMSs double stained for Oct3/4 and cTnT (Figure 2E, a through c; supplemental Video 3). Z-Stack imaging of day-14 BMSs revealed that Oct3/4+ cells concentrate in the core of the BMSs (Figure 2E, a), whereas cTnT+ cells predominately localized to the periphery of the BMSs (Figure 2E, b and c). Detailed analysis revealed the presence of cTnT+ Oct3/4+ cells in the transition from the center to the periphery of the BMSs (Figure 2E, d through f), representing approximately one-fourth of the cardiac myocytes found in the BMSs (cTnT+ cells: Oct3/4+, 2.9±1.9%; Oct3/4–, 10.8±1.9%, of total CCMs cells). To further confirm the key role played by Oct3/4+ cells in cardiac differentiation, targeted Oct3/4 small interfering (siRNA) was performed on day-1 bone marrow cell cultures (Figure 2F). Reduction of Oct3/4 expression at day 3 significantly reduced day-7 expression of the cardiac marker ß-MHC.

BMSs Are Formed by a Heterogeneous Population of Oct3/4+ Stem Cells
To investigate the in vivo origin of the Oct3/4+ population, we examined the bone marrow in situ. Immunostaining confirmed the presence of the Oct3/4+ cells in the bone marrow (Figure 3A, a). The cells were present as isolated cells, mostly localized close to the osteoblastic niche. Consistent with this, immunostaining of freshly isolated bone marrow cells confirmed Oct3/4 expression in rare, isolated cells (0.05±0.03% of cells) (Figure 3A, b).


Figure 3
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  		Figure 3. Cardiomyogenic BMSs are formed by an heterogeneous population of Oct3/4+ stem cells. A, a, Oct3/4-DAB staining of paraffin sections of a 3-month-old mouse femur confirming nuclear expression of Oct3/4+ in bone marrow cells (BM) (arrows) adjacent to the osteoblastic niche (B). Oct3/4+ cells detail (a, inset). Negative isotype control is also shown. b, Immunostaining of day-0 bone marrow cell cytospins for Oct3/4 (fluorescein isothiocyanate [FITC]) and the DNA dye DAPI (blue). Positive control: ES cells. Bars=20 µm. B, Immunostaining of bone marrow cell cytospins (a, c, e, and g) showing that bone marrow–derived Oct3/4+ cells (a, e, and g; FITC; c; Cy3; arrowheads) are also cKit+ (a; FITC), CXCR4+ (c; FITC), Flk1+, FGFR1+ (e; Texas red [TR]), and Flk1+/FGFR1+ (e; TR/Alexa Fluor 350) but Sca1–, CD34– (a; TR; arrows), and CD45– (g; Cy3; arrows). Immunostaining of day-14 BMSs confirmed their phenotypic heterogeneity showing partial expression of cKit (b; FITC; arrowheads), CXCR4 (d; FITC), Flk1, and FGFR1 (f; FITC and TR, arrowhead). CD45+ cells were also observed in the developing BMSs (h). All merged images also show the DNA dye DAPI (blue). Bars=20 µm. C, Graphs showing average percentages of day-0 Oct3/4+ cells coexpressing other biomarkers (3 replicates/group; 1 mouse/2 slides/replicate; 2x105 cells/cytospin slide), and SEM bars.

In vitro analysis of the bone marrow cells revealed significant phenotypic heterogeneity within the Oct3/4+ cell population that only partially overlapped with that of stem cell populations previously described (Figure 3B; supplemental Figure I). Notably, only a fraction of the Oct3/4+ cells costained for the stem cell marker cKit (17.1±1.7%; Figure 3B, a, and Figure 3C), which is also expressed by resident cardiac stem cells21,22 and CXCR4 (58.3±16.7%; Figure 3B, c, and Figure 3C), which has been found in association with Oct3/4 in a population of multipotent bone marrow stem cells previously described.23

Furthermore, the Oct3/4+ cells were negative for the hematopoietic cell markers CD34 and CD45, as well as the mesenchymal stem cell marker Sca1/CD31 (Figure 3B, a and g), which has been previously used to isolate cells for cardiac regeneration.12,23,24 Additional staining showed that a proportion of Oct3/4+ cells also expressed the vascular endothelial growth factor receptor, Flk1/VEGFR2 (21.5±5%) and the fibroblast growth factor receptor 1 (FGFR1) (16.4±12.1%), with only a small proportion of dual positive cells (6.6±4.6%; Figure 3B, e; 3C; supplemental Figure I). Indeed, cells positive for cKit, CXCR4, Flk1, or FGFR1 primarily composed the BMSs (Figure 3B, b, d, and f) highlighted the nonuniform composition of the Oct3/4+ cell aggregates that give rise to cardiac myocytes. Finally, although Oct3/4+ cells did not initially express CD45, CD45+ cells were observed at day 14 at the periphery of developing BMSs, possibly as a result of the differentiation process (Figure 3B, h).

Cardiac Differentiation of Bone Marrow Stem Cells Is Mediated by PDGFR{alpha}/Ligand Signaling Pathways
Immunostaining revealed that day-14 BMSs are surrounded by a distinct population of PDGFR{alpha}+cells (Figure 4A, d) that are Oct3/4–, CD34–, and Sca1+ (Figure 4A, a through c and g). Analysis of phosphorylation patterns confirmed PDGFR{alpha} activation (Figure 4A, e and f). Notably, the predominant phosphorylation on PDGFR{alpha} Tyr720 (Figure 4A, e and g) as compared with Tyr754 (Figure 4A, f) is suggestive of signaling through an {alpha} receptor homodimeric complex rather than a heterodimerization with ß receptor.25,26 Indeed, immunostaining demonstrated a lack of PDGFRß (native or phosphorylated) within or adjacent the BMSs (Figure 4A, h, and Figure 4A, h, inset) and molecular analysis revealed expression of PDGFR{alpha} but not PDGFRß in the bone marrow cell cultures or the BMSs themselves (Figure 4B).


Figure 4
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  		Figure 4. Cardiac differentiation of bone marrow cells is regulated by PDGFR{alpha}/PDGF signaling pathways. A, BMSs formed by Oct3/4+ cells are surrounded by a population of PDGFR{alpha}+/Sca1+ cells that do not express Oct3/4. Immunostaining of day-0 bone marrow cell cytospins showed that PDGFR{alpha}+ cells (a, Texas red [TR]; b and c, fluorescein isothiocyanate [FITC]; arrows) do not express Oct3/4 (FITC; arrowhead) (a) and CD34 (TR) (b) but express Sca1 (TR) (c). Cells expressing PDGFR{alpha} (FITC; arrows) (d) were found at the periphery of day-14 BMSs. Activation of PDGFR{alpha} was confirmed by its phosphorylation on Tyr720 (p720-PDGFR{alpha}; FITC) (e), predominantly observed in the {alpha}{alpha} homodimer. Phosphorylation on Tyr754 (p754-PDGFR{alpha}; FITC) (f), usually associated with the {alpha}ß heterodimer, was only occasionally observed. Colocalization of pTyr720-PDGFR{alpha} (FITC) and Sca1 (Cy3) was also observed at the periphery of day-14 BMSs (arrows) (g). PDGFRß, in its native or phosphorylated form (TR) (h and h, inset) was not expressed in the BMSs. Bar=20 µm. B, RT-PCR results confirming expression of PDGFR{alpha}, but not PDGFRß, in day-7 and -14 BMSs as well as day-0 to day-14 whole bone marrow cell cultures (BM). Controls: heart, positive; no cDNA, negative. Results also showed that incubation at day-0 with PDGFR{alpha}-neutralizing antibody prevented the expression of ß-MHC in day-7 bone marrow cell cultures. No effect was observed in cells incubated with PDGFRß-neutralizing antibody and controls, incubated with no antibodies or control goat serum. C, RT-PCR and quantitative RT-PCR (1 arbitrary unit [AU]=maximal relative value [RV] for each gene) of bone marrow cells cultured under cardiomyogenic conditions showed a direct temporal correlation between the downregulation of Oct3/4 and the upregulation of cardiac-specific genes, {alpha}- and ß-MHC, and of PDGF-A, -B, and -C with affinity for PDGFR{alpha}. No correlation was observed with PDGF-D, which has an exclusive affinity for PDGFRß and remained at basal levels during cardiac differentiation. Data are expressed as mean arbitrary units±SEM. Repeated-measures ANOVA (N=3); *P<0.05, **P≤0.02, ***P≤0.005, ****P≤0.0005.

These data are further supported by our previous observation that PDGFR{alpha}+ cells localize to sites of bone marrow cell–mediated cardiac regeneration10 and further supports the hypothesis that the generation of bone marrow–derived cardiac myocytes from Oct3/4+ stem cells may be regulated by PDGF/PDGFR{alpha} signaling pathway. To test this hypothesis, we assessed the effects of neutralizing antibodies, selectively blocking PDGFR{alpha} or PDGFRß, on the differentiation of bone marrow stem cells cultured for 7 days under cardiomyogenic conditions. Significantly, our results showed that blocking of PDGFR{alpha}, but not PDGFRß, prevented the expression of ß-MHC, used as a marker of cardiac differentiation (Figure 4B).

Gene profiling of Oct3/4, {alpha}- and ß-MHC, PDGF-A, -B, -C, and -D in differentiating bone marrow cells cultures also suggested a correlation between cardiac differentiation of bone marrow stem cells and PDGFR{alpha}/PDGF signaling pathways. RT-PCR and quantitative RT-PCR results (Figure 4C) confirmed the kinetics of the reduction in Oct3/4 expression and the induction of cardiac myocyte genes, similar to what has been previously described for ES cell cardiac myocyte derivation.16 The levels of expression of ß-MHC became detectable at day 5 and dramatically increased at day 7, whereas a-MHC was not detectable until day 7 and peaked at day 14. Notably, induction of PDGF-A, -B, and -C, which can all form dimers (PDGF-AA, -BB, -AB, -CC) that can signal through PDGFR{alpha},27 was inversely correlated with the decrease in Oct3/4 and preceded the expression of ß-MHC and the development of chronotropic activity (Figure 4C). On the contrary, PDGF-D, which interacts only with PDGFRß, remained at basal levels during differentiation. Together with the results of the receptor neutralization studies, these findings suggested that PDGFR{alpha} ligand(s) were central in the differentiation of cardiac myocytes from bone marrow–derived Oct3/4+ cells.

Cardiac Differentiation of Bone Marrow Stem Cells Is Mediated by PDGF-AB/PDGFR{alpha} Signaling Pathways
To better evaluate the significance of the PDGF pathways on cardiomyogenesis, we elected to compare cultures of bone marrow cells isolated from 3 month-old mice with cells isolated from 18-month-old mice, which have an impairment in the induction of PDGF pathways.17 Confocal microscopy demonstrated the expression of both Oct3/4 and PDGFR{alpha} in BMSs from the old (18-month) bone marrow cells that tended to be smaller than BMSs from young (3-month-old) mice (Figure 5A; supplemental Videos 4 and 5). Importantly, Oct3/4 levels in old bone marrow cell cultures were comparable to those observed in young cell cultures (Figure 5B, day 0 and day 7), suggesting that the old cells had the capacity to generate cardiac myocytes in vitro. Cardiac myocyte differentiation as measured by ß-MHC expression, however, revealed a significant impairment in the cardiomyogenic pathways in the aging cell cultures (Figure 5B). Analysis of PDGFR{alpha} and its ligands showed lower levels of expression of PDGFR{alpha} (day 14, P=0.049) and PDGF-B (day 0, P=0.001; day 7, P=0.02; day 14, P=0.001). On the contrary, PDGF-A (day 0 to 14, P≥0.3) and PDGF-C (day 0 to 14, P≥0.1) levels were comparable to those observed in the young. These data suggested that age-associated loss of cardiomyogenic potential is attributable to a specific dysregulation of the PDGF-B/PDGFR{alpha} signaling, rather than impairment in the Oct3/4+ stem cell compartment. To this end, supplementation of PDGF-AB, which has cardioprotective effects on the aging rodent heart,18 was sufficient to restore cardiomyogenic potential of the aging bone marrow cell cultures, as confirmed by the generation of contracting CCMs positive for cTnT (Figure 5C) and the increase in the expression of the cardiac marker ß-MHC (P<0.01), at day 7. Supplementations of old bone marrow cell cultures with PDGF-AA, -BB, and -CC were not able to rescue the differentiation potential of stem cells from the old bone marrow. Interestingly, culture of old (O) bone marrow cells with PDGF-AB for 14 days (O+P) increased expression of PDGFR{alpha} (O: 0.1±0.0; O+P: 0.5±0.1; Y: 0.7±0.2; where Y indicates young), Oct3/4 (O: 0.3±0.1; O+P: 15.0±3.2; Y: 24.0±18.3), and ß-MHC (O: 1.0±0.3; O+P: 3.9±1.7; Y: 2.9±0.3) to levels comparable to those observed in the young (Y) bone marrow cell cultures (O+P versus Y; P>0.05 for all genes). Together these data indicate a specific functional role for the PDGF-AB heterodimer in bone marrow stem cell cardiac differentiation in culture and, moreover, suggest that PDGFR{alpha} and Oct3/4 downregulation might be secondary to PDGF-B dysregulation.


Figure 5
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  		Figure 5. Cardiac differentiation of Oct3/4+ stem cells is mediated by PDGF-B/PDGFR{alpha} signaling pathways. A, Confocal images of day-14 BMSs obtained from 3- and 18-month-old mouse bone marrow cell cultures and stained for Oct3/4 (fluorescein isothiocyanate), PDGFR{alpha} (Cy3), and the DNA dye To-pro-3 (blue). Bars=50 µm. B, Quantitative RT-PCR results comparing the levels of expression of Oct3/4, PDGFR{alpha}, ß-MHC, PDGF-A, PDGF-B, and PDGF-C of bone marrow cell cultures from 3-month-old mice (Y) to those of 18-month-old (O) animals at 3 different time points. Unpaired t test (N=3); *P≤0.05, **P≤0.02, ***P≤0.005. Data are expressed as mean relative value (RV)±SEM. C, a, cTnT+ CCMs were observed at day 14 only in bone marrow cells from 18-month-old mice that were cultured with 10 ng/mL PDGF-AB. b, Quantitative RT-PCR results comparing the levels of expression of ß-MHC at day 7 in bone marrow cell cultures from 18-month-old mice supplemented with 10 ng/mL PDGF-AA, -BB, -CC, AA, or -AB. Data are expressed as mean RV±SEM. Unpaired t test (N=4); **P≤0.01. D, RT-PCR and quantitative RT-PCR results showing that targeted siRNA for PDGF-A and PDGF-B in day-3 bone marrow cell cultures from 3-month-old mice reduced the expression of the cardiac marker ß-MHC at day 7. PDGF-C siRNA had no effect. Controls were treated with scrambled siRNA duplexes. Bars are proportional to the reduction in ß-MHC expression relatively to controls. Values were normalized to relative PDGF levels. Data are expressed as mean AU±SEM. Paired t test (N=4); *P≤0.01, **P≤0.005.

To confirm the importance of PDGF-AB–mediated pathways in the cardiomyogenic differentiation of bone marrow stem cells, we performed targeted PDGF-A, PDGF-B, and PDGF-C siRNA in bone marrow cells of 3-month-old mice. Molecular analysis, at day 7, confirmed that reduction in all the PDGF isoforms but only significant decreases in ß-MHC expression with PDGF-A and -B siRNA (Figure 5D).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Previous reports had shown the ability of purified bone marrow cells to differentiate into a variety of cell types including cardiomyocyte-like cells.10–12,23 Here we focused on the mechanism by which a subpopulation of bone marrow cells can differentiate into cardiac myocytes in vitro. Indeed, we have shown that an autoaggregatory heterogeneous population of bone marrow–derived Oct3/4+ stem cells can differentiate into cardiac myocytes in culture. Moreover, we demonstrated the essential role of PDGF-AB–mediated pathways for this differentiation, revealing that a reversible impairment in trophic support and not the Oct3/4+ stem cell compartment itself underlies the age-related decline in cardiomyogenic capacity.

It has been previously described, by us and others, that bone marrow–derived cells have the ability to differentiate into cells with cardiac myocyte phenotypes.10–12,23 Extending on those observations, we have chronologically and mechanistically assessed the cardiomyogenic capacity of the adult murine bone marrow. Cardiac differentiation was confirmed by the onset of spontaneous chronotropic activity associated with the expression of a range of cardiac genes. Notably, the relative proportion of ß-MHC and the rounded shape of the cells, including those with chronotropic activity is consistent with the development of fetal/neonatal cardiac myocytes and is similar to the cells generated by ES-derived embryoid bodies.28 Indeed, functionality was confirmed as has been previously assessed for differentiating ES cells,29 testing the responsiveness of the bone marrow–derived cardiac myocytes to ß-adrenergic stimulation on multifield electrodes. Together, these experiments enabled us to confirm the in vitro differentiation of bone marrow cells into cardiac myocytes.

Our identification of the BMSs as the source of cardiomyogenic potential facilitated our assessment of this subpopulation of cells. BMSs were morphologically similar to other stem cell aggregates with cardiomyogenic potential, such as ES cell–derived embryoid bodies16 and cardiospheres obtained by culturing resident cardiac stem cells.21,22 Indeed, the cardiac gene profiling and induction of spontaneous chronotropic function in the bone marrow cultures was also comparable to that observed during murine ES cell cardiac differentiation.16,30

Molecular and immunocytochemical analysis of the BMSs confirmed the expression of the stem cell markers Oct3/4,31 nanog,32 and Dppa3,33 which are also expressed by undifferentiated ES cells. The BMSs also expressed prominin-1,34 which was originally considered a progenitor cell marker but has been found, more recently, in adult stem cells in association with the pluripotency markers Oct3/4, Rex-1, Sox-1, and Sox-2,35–37 suggesting it might be expressed in more primitive cell types. Furthermore, downregulation of these genes, and in particular Oct3/4, in the BMSs during cardiac differentiation is consistent with what has been observed in mouse embryoid bodies.16 Moreover, the coexpression of Oct3/4+ and cTnT cells in BMSs and the inverse temporal correlation between Oct3/4 expression and the induction {alpha}- and ß-MHC offers further proof that the cardiac myocytes differentiate from a primitive subpopulation of Oct3/4+ stem cells. Notably, the decrease in Oct3/4 coincided with the upregulation in nestin, a gene that has been shown to be coexpressed with early cardiac markers in ES cells committed to the cardiac lineage.38,39 Indeed, together these findings suggest that bone marrow-cell cardiac differentiation or bone marrow cell differentiation into cardiac myocytes might progress from a more plastic stem cell stage (Oct3/4high/nestinlow) to an intermediate, lineage restricted, cardiac progenitor cell stage (Oct3/4low/nestinhigh).

Like recent studies showing that the cardiomyogenic potential of cardiac stem cells correlates negatively with CD31/Sca140 and positively with cKit,21,22 the present findings suggest that cells of the BMSs may be similar to resident cardiac stem cells. Indeed, the phenotypic similarities we found between bone marrow–derived stem cells and cardiac stem cell populations support the possibility that the bone marrow and the heart may be part of the same stem cell compartment, potentially connected by the systemic circulation. Moreover, the characterization of the Oct3/4+ cells with cardiomyogenic suggested they represent a heterogeneous population of cells that only partially overlapped with stem cell populations previously described. The lack of expression of CD34 and CD45 suggests that they do not belong to the hematopoietic compartment. The Sca1–/cKit+/–/CXCR4+/– phenotype of the cells, in association with their higher levels of Oct3/4 expression, suggests that they are distinct from the Sca1+/cKit–/Oct3/4–/low mesenchymal stem cell population9,41 and the Sca1+/CXCR4+/Oct3/4 multipotent bone marrow stem cell subpopulation.23

In addition to identifying a source of the cardiomyogenic potential in the adult bone marrow, our data offer important mechanistic insights into the differentiation of bone marrow cells into functional cardiac tissue. Expression of Flk1 and FGFR1 in the Oct3/4+ cells, albeit in a heterogeneous pattern, are consistent with the importance of VEGF and basic fibroblast growth factor (FGF-b) in the generation of bone marrow–derived cardiac myocytes.10 In particular, the expression of Flk1 at sites of cardiac differentiation is consistent with the central support role of VEGF and FGF-b pathways in heart tissue development and regeneration.16,42 Moreover, the expression of FGFR1 in Oct3/4+ cells and the requirement for FGF-b for bone marrow cardiomyocyte differentiation parallels the cardiac differentiation of ES cells, where FGF-b/FGFR1 is necessary for the maturation of AFP+ cardiomyogenic endoderm.43

Our molecular and phenotypic analysis also proved that PDGF-AB/PDGFR{alpha} signaling pathway is necessary for cardiac myocyte differentiation. Immunostaining and experiments with neutralizing antibodies confirmed the exclusive involvement of PDGFR{alpha} in cardiac differentiation. Gene profiling results also confirmed the involvement of PDGFR{alpha}-ligand interaction showing that PDGF chains with specific affinity for PDGFR{alpha} (PDGF-A, PDGF-B, PDGF-C) preceded induction of cardiac genes. Moreover, experiments with aging bone marrow cells linked the dysregulation of PDGF-B to the loss of cardiomyogenic potential and showed that this can be reversed only by supplementation of PDGF-AB. The reduction in the expression of cardiac genes observed after PDGF-A and -B siRNA in young bone marrow cell cultures also confirmed the specific involvement of PDGF-AB/PDGFR{alpha} pathway in cardiac differentiation. Our observations are consistent with previous reports showing that PDGF-AB high-affinity binding to PDGFR{alpha} is not only possible, but necessary for significant activation of target cells.44 Furthermore, specific requirement for PDGF-AB/PDGFR{alpha} interaction for cardiac differentiation of bone marrow cells is also consistent with previous data showing that, although PDGFR{alpha} can bind different PDGF isoforms (PDGF-AA,-AB, BB, and CC), each receptor/ligand interaction activates distinct signaling pathways regulating different physiological processes.45 Indeed, such interactions activate distinct PDGFR{alpha} phosphorylation patterns that are then transduced into different downstream signaling pathways. Specifically, we have previously demonstrated that PDGF-AB, but not PDGF-AA, mediates the PDGFR{alpha}-dependent activity of cardiac microvascular endothelial cells in their communication with cardiac myocytes.46 Indeed, such ligand-specific regulation may involve conformational changes in PDGFR{alpha} that can govern signaling cascades, including the recruitment and/or binding of specific cofactors, to promote a microenvironment supportive of the generation of additional cardiac myocytes from generated from bone marrow–derived Oct3/4 cells. Functional advantage of PDGF-AB over PDGF-AA on stem cell differentiation might also be attributable to its stronger mitogenic properties47 conferred by the presence of the B chain, homolog to the simian sarcoma virus (v-sis) transforming protein.48

Together these findings highlight the importance of the local microenvironment in regulating the differentiation potential of the bone marrow–derived Oct3/4+ stem cells and are consistent with data showing the ability of PDGF-AB18 to increase the number of cardiac myocyte bundles in rat models of myocardial infarction and presence of high concentrations of PDGFR{alpha}+ cells in areas of cardiac regeneration.10 Our data are also consistent with previous reports showing involvement of PDGF-B and PDGFR{alpha} in ES cells cardiac differentiation19 in vitro and during development.49

Our dilution studies served to further support the importance of cell–cell interactions in the differentiation of bone marrow–derived cardiac myocytes. Specifically, we showed that cell density is critical to the aggregation and subsequent differentiation of the BMSs from the bone marrow cells in culture. To this end, we speculate that the cardioplasticity of the bone marrow cells requires a heterogeneous population of Oct3/4+ cells that can interact with PDGFR{alpha} cells to promote the generation of cardiac myocytes. Thus, it is likely that the isolation of individual Oct3/4+ cells in the bone marrow may be essential in preserving the plasticity and/or quiescence of this subpopulation of stem cells. Indeed, we speculate that paracrine/juxtacrine pathways in the bone marrow may govern the integrity of the Oct3/4+ cells, potentially preventing their aggregation and possible differentiation in the bone marrow itself. Outside of the bone marrow, such as in an in vitro culture system, our results have shown that the Oct3/4+ cells can spontaneously aggregate and differentiate. Moreover, we demonstrated that both Oct3/4 expression and self-aggregation are preserved in the aging bone marrow and that age-associated impairment in cardiac regenerative potential50 is determined by a reversible dysregulation of the PDGF-AB/PDGFR{alpha} signaling pathways. This result is in contrast with previous reports that linked the age-related loss of cardiomyogenic potential to a reduction of a specific bone marrow stem cell population (CXCR4+/Oct3/4+/Sca1+).23 This discrepancy can be explained by the different experimental approaches. Previous studies focused on the purification and characterization of multipotent stem cells, whereas the present study focused on the mechanisms of cardiac differentiation. For this purpose, unfractionated bone marrow cells were used to preserve and study the complex interactions of heterogeneous populations of cells involved in cardiac differentiation, which may not have been previously appreciated.

Notably, the potential of bone marrow–derived Oct3/4+ cells to differentiate into cells other than cardiac myocytes was not the focus of this present work and the mechanisms governing their differentiation into noncardiac cells remains to be defined. Previous studies have demonstrated the significance of Oct3/4 in the extended nuclear plasticity of stem cells.51,52 Notably, although the BMS Oct3/4 level was approximately one-tenth that of undifferentiated ES cells, it was significantly higher compared with the expression levels reported in multipotent adult stem cells (MAPCs),9,41 suggesting that the murine BMSs may have lineage plasticity similar to that observed in the MAPCs. To this end, we speculate that the noncardiac differentiation of the BMS Oct3/4+ cells may be dependent paracrine/juxtacrine communications that are analogous the PDGF pathways that drive cardiac myocyte generation. Indeed, we hypothesize that the identification of such cell–cell interactions in other tissue microenvironments may enable the development of approached to direct the plasticity of the BMS Oct3/4+ cells.

In conclusion, the present data identify a cardiac stem cell population in the bone marrow and the paracrine/juxtacrine mechanisms that regulate its differentiation. Moreover, these data suggest that strategies aimed at targeting the bone marrow–derived Oct3/4+ cells and that their interactions of PDGF-AB with PDGFR{alpha}+ cells may be used to enhance cardiac regeneration, particularly in older individuals.


*    Acknowledgments
 
We thank Dr Peter Okin for assistance with statistical analyses.

Sources of Funding

This work was supported in part by the NIH (AG19738 and AG20918) and a Pfizer/American Federation in Aging Research Innovations in Aging Research Award.

Disclosures

None.


*    Footnotes
 
Original received April 19, 2006; first resubmission received September 6, 2006; second resubmission received October 27, 2006; accepted November 9, 2006.


*    References
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 up arrowIntroduction
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 *References
 

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