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1.  Role of the ATP- binding cassette transporter Abcg2 in the phenotype and function of cardiac side population cells 
Circulation research  2008;103(8):825-835.
Recently, the side population (SP) phenotype has been introduced as a reliable marker to identify subpopulations of cells with stem/progenitor cell properties in various tissues. We and others have identified SP cells from post-mitotic tissues, including adult myocardium, in which they have been suggested to contribute to cellular regeneration following injury. SP cells are identified and characterized by a unique efflux of Hoechst 33342 dye. Abcg2 belongs to the ATP-binding cassette (ABC) transporter superfamily and constitutes the molecular basis for the dye efflux, hence the SP phenotype, in hematopoietic stem cells. While Abcg2 is also expressed in cardiac SP (cSP) cells, its role in regulating the SP phenotype and function of cSP cells is unknown. Herein, we demonstrate that regulation of the SP phenotype in cSP cells occurs in a dynamic, age-dependent fashion, with Abcg2 as the molecular determinant of the cSP phenotype in the neonatal heart and another ABC transporter, Mdr1, as the main contributor to the SP phenotype in the adult heart. Using loss- and gain-of-function experiments we find that Abcg2 tightly regulates cell fate and function. Adult cSP cells isolated from mice with genetic ablation of Abcg2, exhibit blunted proliferation capacity and augmented cell death. Conversely, overexpression of Abcg2 is sufficient to enhance cell proliferation, though with a limitation of cardiomyogenic differentiation. In summary, for the first time, we reveal a functional role for Abcg2 in modulating the proliferation, differentiation, and survival of adult cSP cells that goes beyond its distinct role in Hoechst dye efflux.
doi:10.1161/CIRCRESAHA.108.174615
PMCID: PMC3115759  PMID: 18787193
Abcg2; Cardiomyocytes; Cell death; Contractility; Differentiation; Mdr1; Progenitor cells; Proliferation; SP cells; Stem cells
2.  Cardiac side population cells have a potential to migrate and differentiate into cardiomyocytes in vitro and in vivo 
The Journal of Cell Biology  2007;176(3):329-341.
Side population (SP) cells, which can be identified by their ability to exclude Hoechst 33342 dye, are one of the candidates for somatic stem cells. Although bone marrow SP cells are known to be long-term repopulating hematopoietic stem cells, there is little information about the characteristics of cardiac SP cells (CSPs). When cultured CSPs from neonatal rat hearts were treated with oxytocin or trichostatin A, some CSPs expressed cardiac-specific genes and proteins and showed spontaneous beating. When green fluorescent protein–positive CSPs were intravenously infused into adult rats, many more (∼12-fold) CSPs were migrated and homed in injured heart than in normal heart. CSPs in injured heart differentiated into cardiomyocytes, endothelial cells, or smooth muscle cells (4.4%, 6.7%, and 29% of total CSP-derived cells, respectively). These results suggest that CSPs are intrinsic cardiac stem cells and involved in the regeneration of diseased hearts.
doi:10.1083/jcb.200603014
PMCID: PMC2063959  PMID: 17261849
3.  Cardiac Side Population Cells: Moving toward the Center Stage in Cardiac Regeneration 
Circulation research  2012;110(10):1355-1363.
Over the past decade, extensive work in animal models and humans has identified the presence of progenitor cells within the adult heart, capable of cardiomyogenic differentiation and likely contributors to cardiomyocyte turnover during normal development and disease. Among the identified cardiac progenitor cells, there is a distinct subpopulation, termed “side population” (SP) progenitor cells, which are isolated by their unique ability to efflux DNA binding dyes through ATP-binding cassette transporter. This review highlights the literature on the isolation, characterization and functional relevance of CSP cells. We review the initial discovery of cardiac SP (CSP) cells in adult myocardium, as well as their capacity for functional cardiomyogenic differentiation and role in cardiac regeneration following myocardial injury. Finally, we discuss recent advances in understanding the molecular regulators of cardiac SP cell proliferation and differentiation, as well as likely future areas of investigation required to realize the goal of effective cardiac regeneration.
doi:10.1161/CIRCRESAHA.111.243014
PMCID: PMC3412159  PMID: 22581921
Adult stem cells; Cardiac progenitor cells; Cardiogenesis; Cardiovascular disease; Regeneration
4.  Wnt signaling exerts an anti-proliferative effect on adult cardiac progenitor cells via IGFBP3 
Circulation Research  2011;109(12):1363-1374.
Rationale
Recent work in animal models and humans has demonstrated the presence of organ-specific progenitor cells required for the regenerative capacity of the adult heart. In response to tissue injury, progenitor cells differentiate into specialized cells, while their numbers are maintained through mechanisms of self-renewal. The molecular cues that dictate the self-renewal of adult progenitor cells in the heart, however, remain unclear.
Objective
Herein, we investigate the role of canonical Wnt signaling on adult cardiac side population (CSP) cells under physiological and disease conditions.
Methods and Results
CSP cells isolated from C57BL/6J mice were utilized to study the effects of canonical Wnt signaling on their proliferative capacity. The proliferative capacity of CSP cells was also tested following injection of recombinant Wnt3a protein (r-Wnt3a) in the left ventricular free wall. Wnt signaling was found to decrease the proliferation of adult CSP cells, both in vitro and in vivo, through suppression of cell cycle progression. Wnt stimulation exerted its anti-proliferative effects through a previously unappreciated activation of insulin-like growth factor binding protein 3 (IGFBP3), which requires intact IGF binding site for its action. Moreover, injection of r-Wnt3a following myocardial infarction in mice showed that Wnt signaling limits CSP cell renewal, blocks endogenous cardiac regeneration and impairs cardiac performance, highlighting the importance of progenitor cells in maintaining tissue function after injury.
Conclusions
Our study identifies canonical Wnt signaling and the novel downstream mediator, IGFBP3, as key regulators of adult cardiac progenitor self-renewal in physiological and pathological states.
doi:10.1161/CIRCRESAHA.111.250282
PMCID: PMC3384997  PMID: 22034491
cardiac side population cells; Wnt signaling; cardiac regeneration; stem cells; proliferation
5.  TGFβ-Dependent Epithelial-to-Mesenchymal Transition Is Required to Generate Cardiospheres from Human Adult Heart Biopsies 
Stem Cells and Development  2012;21(17):3081-3090.
Autologous cardiac progenitor cells (CPCs) isolated as cardiospheres (CSps) represent a promising candidate for cardiac regenerative therapy. A better understanding of the origin and mechanisms underlying human CSps formation and maturation is undoubtedly required to enhance their cardiomyogenic potential. Epithelial-to-mesenchymal transition (EMT) is a key morphogenetic process that is implicated in the acquisition of stem cell-like properties in different adult tissues, and it is activated in the epicardium after ischemic injury to the heart. We investigated whether EMT is involved in the formation and differentiation of human CSps, revealing that an up-regulation of the expression of EMT-related genes accompanies CSps formation that is relative to primary explant-derived cells and CSp-derived cells grown in a monolayer. EMT and CSps formation is enhanced in the presence of transforming growth factor β1 (TGFβ1) and drastically blocked by the type I TGFβ-receptor inhibitor SB431452, indicating that TGFβ-dependent EMT is essential for the formation of these niche-like 3D-multicellular clusters. Since TGFβ is activated in the myocardium in response to injury, our data suggest that CSps formation mimics an adaptive mechanism that could potentially be enhanced to increase in vivo or ex vivo regenerative potential of adult CPCs.
doi:10.1089/scd.2012.0277
PMCID: PMC4146498  PMID: 22765842
6.  Abcg2 Regulates Cell Cycle Progression and Asymmetric Division in Mouse Cardiac Side Population Progenitor Cells 
Circulation research  2012;112(1):27-34.
Rationale
Following cardiac injury, cardiac progenitor cells are acutely reduced, and are replenished in part by regulated self-renewal and proliferation, which occurs through symmetric and asymmetric cellular division. Understanding the molecular cues controlling progenitor cell self-renewal and lineage commitment is critical towards harnessing these cells for therapeutic regeneration. We have previously found that the cell surface ATP binding cassette (ABC)-transporter, Abcg2, influences the proliferation of cardiac side population (CSP) progenitor cells, though through unclear mechanisms.
Objective
To determine the role of Abcg2 on cell cycle progression and mode of division in mouse CSP cells.
Methods and Results
Herein, using CSP cells isolated from wild-type and Abcg2-knockout mice, we find that Abcg2 regulates G1-S cell cycle transition by FUCCI cell cycle indicators, cell cycle-focused gene expression arrays and confocal live cell fluorescent microscopy. Moreover, we find that modulation of cell cycle results in transition from symmetric to asymmetric cellular division in CSP cells lacking Abcg2.
Conclusions
Abcg2 modulates CSP cell cycle progression and asymmetric cell division, establishing a mechanistic link between this surface transporter and cardiac progenitor cell function. Greater understanding of progenitor cell biology, and in particular the regulation of resident progenitor cell homeostasis, is vital for guiding the future development of cell-based therapies for cardiac regeneration.
doi:10.1161/CIRCRESAHA.111.300010
PMCID: PMC3959170  PMID: 23136123
ABC transporter; cardiac side population cells; asymmetric division; adult stem cell; cell cycle
7.  Urotensin II inhibited the proliferation of cardiac side population cells in mice during pressure overload by JNK-LRP6 signalling 
Cardiac side population cells (CSPs) are promising cell resource for the regeneration in diseased heart as intrinsic cardiac stem cells. However, the relative low ratio of CSPs in the heart limited the ability of CSPs to repair heart and improve cardiac function effectively under pathophysiological condition. Which factors limiting the proliferation of CSPs in diseased heart are unclear. Here, we show that urotensin II (UII) regulates the proliferation of CSPs by c-Jun N-terminal kinase (JNK) and low density lipoprotein receptor-related protein 6 (LRP6) signalling during pressure overload. Pressure overload greatly upregulated UII level in plasma, UII receptor (UT) antagonist, urantide, promoted CSPs proliferation and improved cardiac dysfunction during chronic pressure overload. In cultured CSPs subjected to mechanical stretch (MS), UII significantly inhibited the proliferation by UT. Nanofluidic proteomic immunoassay showed that it is the JNK activation, but not the extracellular signal-regulated kinase signalling, that involved in the UII-inhibited- proliferation of CSPs during pressure overload. Further analysis in vitro indicated UII-induced-phospho-JNK regulates phosphorylation of LRP6 in cultured CSPs after MS, which is important in the inhibitory effect of UII on the CSPs during pressure overload. In conclusion, UII inhibited the proliferation of CSPs by JNK/LRP6 signalling during pressure overload. Pharmacological inhibition of UII promotes CSPs proliferation in mice, offering a possible therapeutic approach for cardiac failure induced by pressure overload.
doi:10.1111/jcmm.12230
PMCID: PMC4119391  PMID: 24447593
Urotensin II; CSPs; proliferation; JNK; LRP6
8.  Leukemia Inhibitory Factor Enhances Endogenous Cardiomyocyte Regeneration after Myocardial Infarction 
PLoS ONE  2016;11(5):e0156562.
Cardiac stem cells or precursor cells regenerate cardiomyocytes; however, the mechanism underlying this effect remains unclear. We generated CreLacZ mice in which more than 99.9% of the cardiomyocytes in the left ventricular field were positive for 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal) staining immediately after tamoxifen injection. Three months after myocardial infarction (MI), the MI mice had more X-gal-negative (newly generated) cells than the control mice (3.04 ± 0.38/mm2, MI; 0.47 ± 0.16/mm2, sham; p < 0.05). The cardiac side population (CSP) cell fraction contained label-retaining cells, which differentiated into X-gal-negative cardiomyocytes after MI. We injected a leukemia inhibitory factor (LIF)-expression construct at the time of MI and identified a significant functional improvement in the LIF-treated group. At 1 month after MI, in the MI border and scar area, the LIF-injected mice had 31.41 ± 5.83 X-gal-negative cardiomyocytes/mm2, whereas the control mice had 12.34 ± 2.56 X-gal-negative cardiomyocytes/mm2 (p < 0.05). Using 5-ethynyl-2'-deoxyurinide (EdU) administration after MI, the percentages of EdU-positive CSP cells in the LIF-treated and control mice were 29.4 ± 2.7% and 10.6 ± 3.7%, respectively, which suggests that LIF influenced CSP proliferation. Moreover, LIF activated the Janus kinase (JAK)signal transducer and activator of transcription (STAT), mitogen-activated protein kinase/extracellular signal-regulated (MEK)extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K)–AKT pathways in CSPs in vivo and in vitro. The enhanced green fluorescent protein (EGFP)-bone marrow-chimeric CreLacZ mouse results indicated that LIF did not stimulate cardiogenesis via circulating bone marrow-derived cells during the 4 weeks following MI. Thus, LIF stimulates, in part, stem cell-derived cardiomyocyte regeneration by activating cardiac stem or precursor cells. This approach may represent a novel therapeutic strategy for cardiogenesis.
doi:10.1371/journal.pone.0156562
PMCID: PMC4881916  PMID: 27227407
9.  PDGFRα demarcates the cardiogenic clonogenic Sca1+ stem/progenitor cell in adult murine myocardium 
Nature Communications  2015;6:6930.
Cardiac progenitor/stem cells in adult hearts represent an attractive therapeutic target for heart regeneration, though (inter)-relationships among reported cells remain obscure. Using single-cell qRT–PCR and clonal analyses, here we define four subpopulations of cardiac progenitor/stem cells in adult mouse myocardium all sharing stem cell antigen-1 (Sca1), based on side population (SP) phenotype, PECAM-1 (CD31) and platelet-derived growth factor receptor-α (PDGFRα) expression. SP status predicts clonogenicity and cardiogenic gene expression (Gata4/6, Hand2 and Tbx5/20), properties segregating more specifically to PDGFRα+ cells. Clonal progeny of single Sca1+ SP cells show cardiomyocyte, endothelial and smooth muscle lineage potential after cardiac grafting, augmenting cardiac function although durable engraftment is rare. PDGFRα− cells are characterized by Kdr/Flk1, Cdh5, CD31 and lack of clonogenicity. PDGFRα+/CD31− cells derive from cells formerly expressing Mesp1, Nkx2-5, Isl1, Gata5 and Wt1, distinct from PDGFRα−/CD31+ cells (Gata5 low; Flk1 and Tie2 high). Thus, PDGFRα demarcates the clonogenic cardiogenic Sca1+ stem/progenitor cell.
Adult cardiac progenitor/stem cells (CPSCs) possess valuable potential for heart repair that is limited by the elusiveness of these cells. Here Noseda et al. refine the definition of murine CPSCs producing stem cell antigen 1 (Sca1), mapping the cardiogenic signature and clonogenicity to the subgroup of Sca1+ cells expressing PDGFRα.
doi:10.1038/ncomms7930
PMCID: PMC4479024  PMID: 25980517
10.  Calorie Restriction Increases Muscle Mitochondrial Biogenesis in Healthy Humans 
PLoS Medicine  2007;4(3):e76.
Background
Caloric restriction without malnutrition extends life span in a range of organisms including insects and mammals and lowers free radical production by the mitochondria. However, the mechanism responsible for this adaptation are poorly understood.
Methods and Findings
The current study was undertaken to examine muscle mitochondrial bioenergetics in response to caloric restriction alone or in combination with exercise in 36 young (36.8 ± 1.0 y), overweight (body mass index, 27.8 ± 0.7 kg/m2) individuals randomized into one of three groups for a 6-mo intervention: Control, 100% of energy requirements; CR, 25% caloric restriction; and CREX, caloric restriction with exercise (CREX), 12.5% CR + 12.5% increased energy expenditure (EE). In the controls, 24-h EE was unchanged, but in CR and CREX it was significantly reduced from baseline even after adjustment for the loss of metabolic mass (CR, −135 ± 42 kcal/d, p = 0.002 and CREX, −117 ± 52 kcal/d, p = 0.008). Participants in the CR and CREX groups had increased expression of genes encoding proteins involved in mitochondrial function such as PPARGC1A, TFAM, eNOS, SIRT1, and PARL (all, p < 0.05). In parallel, mitochondrial DNA content increased by 35% ± 5% in the CR group (p = 0.005) and 21% ± 4% in the CREX group (p < 0.004), with no change in the control group (2% ± 2%). However, the activity of key mitochondrial enzymes of the TCA (tricarboxylic acid) cycle (citrate synthase), beta-oxidation (beta-hydroxyacyl-CoA dehydrogenase), and electron transport chain (cytochrome C oxidase II) was unchanged. DNA damage was reduced from baseline in the CR (−0.56 ± 0.11 arbitrary units, p = 0.003) and CREX (−0.45 ± 0.12 arbitrary units, p = 0.011), but not in the controls. In primary cultures of human myotubes, a nitric oxide donor (mimicking eNOS signaling) induced mitochondrial biogenesis but failed to induce SIRT1 protein expression, suggesting that additional factors may regulate SIRT1 content during CR.
Conclusions
The observed increase in muscle mitochondrial DNA in association with a decrease in whole body oxygen consumption and DNA damage suggests that caloric restriction improves mitochondrial function in young non-obese adults.
Anthony Civitarese and colleagues observed an increase in mitochondrial DNA in muscle and a decrease in whole body oxygen consumption in healthy adults who underwent caloric restriction.
Editors' Summary
Background.
Life expectancy (the average life span) greatly increased during the 20th century in most countries, largely due to improved hygiene, nutrition, and health care. One possible approach to further increase human life span is “caloric restriction.” A calorie-restricted diet provides all the nutrients necessary for a healthy life but minimizes the energy (calories) supplied in the diet. This type of diet increases the life span of mice and delays the onset of age-related chronic diseases such as heart disease and stroke. There are also hints that people who eat a calorie-restricted diet might live longer than those who overeat. People living in Okinawa, Japan, have a lower energy intake than the rest of the Japanese population and an extremely long life span. In addition, calorie-restricted diets beneficially affect several biomarkers of aging, including decreased insulin sensitivity (a precursor to diabetes). But how might caloric restriction slow aging? A major factor in the age-related decline of bodily functions is the accumulation of “oxidative damage” in the body's proteins, fats, and DNA. Oxidants—in particular, chemicals called “free radicals”—are produced when food is converted to energy by cellular structures called mitochondria. One theory for how caloric restriction slows aging is that it lowers free-radical production by inducing the formation of efficient mitochondria.
Why Was This Study Done?
Despite hints that caloric restriction might have similar effects in people as in rodents, there have been few well-controlled studies on the effect of good quality calorie-reduced diets in healthy people. It is also unknown whether an energy deficit produced by increasing physical activity while eating the same amount of food has the same effects as caloric restriction. Finally, it is unclear how caloric restriction alters mitochondrial function. The Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE) organization is investigating the effect of caloric restriction interventions on physiology, body composition, and risk factors for age-related diseases. In this study, the researchers have tested the hypothesis that short-term caloric deficit (with or without exercise) increases the efficiency of mitochondria in human muscle.
What Did the Researchers Do and Find?
The researchers enrolled 36 healthy overweight but non-obese young people into their study. One-third of them received 100% of their energy requirements in their diet; the caloric restriction (CR) group had their calorie intake reduced by 25%; and the caloric restriction plus exercise (CREX) group had their calorie intake reduced by 12.5% and their energy expenditure increased by 12.5%. The researchers found that a 25% caloric deficit for six months, achieved by diet alone or by diet plus exercise, decreased 24-hour whole body energy expenditure (i.e., overall calories burned for body function), which suggests improved mitochondrial function. Their analysis of genes involved in mitochondria formation indicated that CR and CREX both increased the number of mitochondria in skeletal muscle. Both interventions also reduced the amount of DNA damage—a marker of oxidative stress—in the participants' muscles.
What Do These Findings Mean?
These results indicate that a short-term caloric deficit, whether achieved by diet or by diet plus exercise, induces the formation of “efficient mitochondria” in people just as in rodents. The induction of these efficient mitochondria in turn reduces oxidative damage in skeletal muscles. Consequently, this adaptive response to caloric restriction might have the potential to slow aging and increase longevity in humans as in other animals. However, this six-month study obviously provides no direct evidence for this, and, by analogy with studies in rodents, an increase in longevity might require lifelong caloric restriction. The results here suggest that even short-term caloric restriction can produce beneficial physiological changes, but more research is necessary before it becomes clear whether caloric restriction should be recommended to healthy individuals.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040076.
The CALERIE (Comprehensive Assessment of Long-Term Effects of Reducing Intake of Energy) Web site contains information on the study and how to participate
American Federation for Aging Research includes information on aging with pages on the biology of aging and on caloric restriction
The Okinawa Centenarian Study is a population-based study on long-lived elderly people in Okinawa, Japan
US Government information on nutrition
MedlinePlus encyclopedia pages on diet and calories
The Calorie Restriction Society, a nonprofit organization that provides information on life span and caloric restriction
Wikipedia pages on calorie restriction and on mitochondria (note: Wikipedia is an online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0040076
PMCID: PMC1808482  PMID: 17341128
11.  Identification of a nonameric H-2Kk-restricted CD8+ cytotoxic T lymphocyte epitope on the Plasmodium falciparum circumsporozoite protein. 
Infection and Immunity  1995;63(5):1955-1959.
Class I-restricted CD8+ cytotoxic T lymphocytes (CTL) against the circumsporozoite protein (CSP) protect mice against the rodent malaria parasite, Plasmodium yoelii, and vaccines designed to produce protective CTL against the P. falciparum CSP (PfCSP) are under development. Humans and B10.BR (H-2k) mice have been shown to have CD8+ CTL activity against a 23-amino-acid region of the PfCSP (residues 368 to 390 from the PfCSP 7G8 sequence) that is too long to bind directly to class I major histocompatibility complex molecules. To identify within this 23-amino-acid peptide a shorter peptide that binds to an H-2k class I major histocompatibility molecule, a primarily CD8+ (97.8%) T-cell line (PfCSP TCL.1) was produced by immunizing B10.BR mice with recombinant vaccinia virus expressing the PfCSP and stimulating in vitro spleen cells from these immunized mice with L cells transfected with the PfCSP gene (LPF cells). PfCSP TCL.1 lysed LPF cells and L cells pulsed with peptide PfCSP 7G8 368-390. When 15 overlapping nonamer peptides spanning the 368 to 390 sequence were tested, only one peptide, PfCSP 7G8 375-383 (Y E N D I E K K I), which includes an H-2Kk-binding motif, E at amino acid residue 2, and I at residue 9, sensitized targets for lysis by PfCSP TCL.1. Furthermore, a 10(3)- to 10(4)-fold lower concentration of the nonamer than that of the 23-amino-acid peptide was required to sensitize target cells for lysis by PfCSP TCL.1. Presentation by H-2Kk was demonstrated by using 3T3 fibroblast cells transfected with the murine H-2Kk or H-2Dk genes, and only the H-2Kk transfectants were lysed by PfCSP TCL.1 after incubation with peptide PfCSP 7G8 375-383. Binding to H-2Kk was confirmed by competitive inhibition of binding of labelled peptides to affinity-purified Kk molecules. Substitution of the anchor amino acid residue, E, at position 2 with A dramatically reduced binding to Kk and eliminated the capacity of the peptide to sensitize target cells for killing. Variation of non-anchor residues did not markedly reduce binding to Kk but in some cases eliminated the capacity of the peptide to sensitize targets for cytolysis by PfCSP TCL.1, presumably by eliminating T-cell receptor-binding sites. These data suggest that similar studies with human T cells will be required for optimal development of peptide-based vaccines designed to produce protective class I-restricted CD8+ CTL against the PfCSP in humans.
PMCID: PMC173249  PMID: 7537251
12.  Optimised Protocols for the Identification of the Murine Cardiac Side Population 
Stem cell reviews  2013;9(5):731-739.
Cardiac side population (CSP) cells, defined by their ability to efflux the vital dye Hoechst 33342, have been identified as putative cardiac stem cells based on their potential to give rise to both cardiomyocytes and endothelial cells. The CSP phenotype relies on an active metabolic pathway and cell viability to identify a rare population of cells and therefore technical differences in the CSP staining protocol can lead to inconsistent results and discrepancies between studies. Here we describe an established protocol for CSP identification and have optimised a protocol for CSP analysis utilizing an automated cardiac digestion procedure using gentleMACs dissociation and Hoechst 33342 staining followed by dual wavelength flow cytometric analysis.
doi:10.1007/s12015-013-9440-9
PMCID: PMC4629417  PMID: 23619929
Cardiac; Side population; Abcg2; Fumitremorgin C; Stem cell
13.  Intracoronary Delivery of Self-Assembling Heart-Derived Microtissues (“Cardiospheres”) for Prevention of Adverse Remodeling in a Pig Model of Convalescent Myocardial Infarction 
Circulation. Cardiovascular interventions  2015;8(5):10.1161/CIRCINTERVENTIONS.115.002391 e002391.
Background
Preclinical studies in rodents and pigs indicate that the self-assembling microtissues known as cardiospheres (CSp) may be more effective than dispersed CSp-derived cells (CDCs). However, the more desirable intracoronary (IC) route has been assumed to be unsafe for CSp delivery: CSp are large (30-150 μm), raising concerns about likely micro-embolization. We questioned these negative assumptions by evaluating the safety and efficacy of optimized IC delivery of CSp in a porcine model of convalescent MI.
Methods and Results
First, we standardized the size of CSp by modifying culture conditions. Then, dosage was determined by infusing escalating doses of CSp in the LAD of naïve pigs, looking for acute adverse effects. Finally in a randomized efficacy study, 14 mini-pigs received allogeneic CSp (1.3×106) or vehicle one month following MI. Animals underwent MRI before infusion and 1 month later to assess left ventricular (LV) ejection fraction (EF), scar mass and viable mass. In the dosing study, we did not observe any evidence of micro-embolization after CSp infusion. In the post-MI study, CSp preserved LV function, reduced scar mass and increased viable mass whereas placebo did not. Moreover, CSp decreased collagen content, and increased vessel densities and myocardial perfusion. Importantly, IC CSp decreased LV end diastolic pressure and increased cardiac output.
Conclusions
Intracoronary delivery of CSp is safe. Intracoronary CSp are also remarkably effective in decreasing scar, halting adverse remodeling, increasing myocardial perfusion and improving hemodynamic status post-MI in pigs. Thus, CSp may be viable therapeutic candidates for IC infusion in selected myocardial disorders.
doi:10.1161/CIRCINTERVENTIONS.115.002391
PMCID: PMC4428690  PMID: 25953823
stem cell; cardiospheres; myocardial infarction; adverse remodeling
14.  Vascular Calcifying Progenitor Cells Possess Bidirectional Differentiation Potentials 
PLoS Biology  2013;11(4):e1001534.
Calcifying progenitor cells in blood vessels have the potential to differentiate into cells that either promote calcium accumulation or reverse accumulation, and treatment with PPAR? can shift the direction of this differentiation.
Vascular calcification is an advanced feature of atherosclerosis for which no effective therapy is available. To investigate the modulation or reversal of calcification, we identified calcifying progenitor cells and investigated their calcifying/decalcifying potentials. Cells from the aortas of mice were sorted into four groups using Sca-1 and PDGFRα markers. Sca-1+ (Sca-1+/PDGFRα+ and Sca-1+/PDGFRα−) progenitor cells exhibited greater osteoblastic differentiation potentials than Sca-1− (Sca-1−/PDGFRα+ and Sca-1−/PDGFRα−) progenitor cells. Among Sca-1+ progenitor populations, Sca-1+/PDGFRα− cells possessed bidirectional differentiation potentials towards both osteoblastic and osteoclastic lineages, whereas Sca-1+/PDGFRα+ cells differentiated into an osteoblastic lineage unidirectionally. When treated with a peroxisome proliferator activated receptor γ (PPARγ) agonist, Sca-1+/PDGFRα− cells preferentially differentiated into osteoclast-like cells. Sca-1+ progenitor cells in the artery originated from the bone marrow (BM) and could be clonally expanded. Vessel-resident BM-derived Sca-1+ calcifying progenitor cells displayed nonhematopoietic, mesenchymal characteristics. To evaluate the modulation of in vivo calcification, we established models of ectopic and atherosclerotic calcification. Computed tomography indicated that Sca-1+ progenitor cells increased the volume and calcium scores of ectopic calcification. However, Sca-1+/PDGFRα− cells treated with a PPARγ agonist decreased bone formation 2-fold compared with untreated cells. Systemic infusion of Sca-1+/PDGFRα− cells into Apoe−/− mice increased the severity of calcified atherosclerotic plaques. However, Sca-1+/PDGFRα− cells in which PPARγ was activated displayed markedly decreased plaque severity. Immunofluorescent staining indicated that Sca-1+/PDGFRα− cells mainly expressed osteocalcin; however, activation of PPARγ triggered receptor activator for nuclear factor-κB (RANK) expression, indicating their bidirectional fate in vivo. These findings suggest that a subtype of BM-derived and vessel-resident progenitor cells offer a therapeutic target for the prevention of vascular calcification and that PPARγ activation may be an option to reverse calcification.
Author Summary
Atherosclerosis involves hardening of the arteries and can lead to heart disease. Calcium accumulation in blood vessels contributes to this process, and this process is regulated by cells that promote calcium accumulation (osteoblasts) and cells that reverse the accumulation (osteoclasts). In this study, we show that vascular calcifying progenitor cells in the blood vessel have the potential to become either osteoblasts or osteoclasts, and that a drug can push these cells towards becoming osteoclasts instead of osteoblasts. Progenitor cells that express both Sca-1 and PDGFRα cell surface proteins were more committed to differentiate into osteoblasts, while cells that only expressed Sca-1 could differentiate into osteoblasts or osteoclasts in a bidirectional manner. Moreover, treatment with a PPARγ agonist could shift the direction of differentiation of Sca-1+/PDGFRα− progenitor cells toward osteoclast-like cells, whereas it cannot influence the fates of Sca-1+/PDGFRα+ progenitors. These results offer new therapeutic targets for reversing calcium accumulation in blood vessels.
doi:10.1371/journal.pbio.1001534
PMCID: PMC3621676  PMID: 23585735
15.  Pneumococcal Competence Coordination Relies on a Cell-Contact Sensing Mechanism 
PLoS Genetics  2016;12(6):e1006113.
Bacteria have evolved various inducible genetic programs to face many types of stress that challenge their growth and survival. Competence is one such program. It enables genetic transformation, a major horizontal gene transfer process. Competence development in liquid cultures of Streptococcus pneumoniae is synchronized within the whole cell population. This collective behavior is known to depend on an exported signaling Competence Stimulating Peptide (CSP), whose action generates a positive feedback loop. However, it is unclear how this CSP-dependent population switch is coordinated. By monitoring spontaneous competence development in real time during growth of four distinct pneumococcal lineages, we have found that competence shift in the population relies on a self-activated cell fraction that arises via a growth time-dependent mechanism. We demonstrate that CSP remains bound to cells during this event, and conclude that the rate of competence development corresponds to the propagation of competence by contact between activated and quiescent cells. We validated this two-step cell-contact sensing mechanism by measuring competence development during co-cultivation of strains with altered capacity to produce or respond to CSP. Finally, we found that the membrane protein ComD retains the CSP, limiting its free diffusion in the medium. We propose that competence initiator cells originate stochastically in response to stress, to form a distinct subpopulation that then transmits the CSP by cell-cell contact.
Author Summary
Development of competence for genetic transformation by cultures of pneumococcal cells has been considered till now as a classic example of quorum sensing, whereby a culture attaining a sufficient cell density detects a diffusible signaling molecule (in this case, Competence-Stimulating Peptide (CSP)) and switches en masse to a distinct physiological state. We find that the competence shift is dictated not by cell density but by growth for a time allowing emergence of a competence-initiator sub-population, and spreads by transmission of CSP through cell contact. This behaviour reflects the survival benefits of allowing subsets of the population to respond to environmental stress by generating signalling capacity, which prepares the entire population for a rapid and appropriate response to threatening conditions.
doi:10.1371/journal.pgen.1006113
PMCID: PMC4927155  PMID: 27355362
16.  Progenitor cells isolated from the human heart: a potential cell source for regenerative therapy 
Netherlands Heart Journal  2008;16(5):163-169.
Background
In recent years, resident cardiac progenitor cells have been identified in, and isolated from the rodent heart. These cells show the potential to form cardiomyocytes, smooth muscle cells, and endothelial cells in vitro and in vivo and could potentially be used as a source for cardiac repair. However, previously described cardiac progenitor cell populations show immature development and need co-culture with neonatal rat cardiomyocytes in order to differentiate in vitro. Here we describe the localisation, isolation, characterisation, and differentiation of cardiomyocyte progenitor cells (CMPCs) isolated from the human heart.
Methods
hCMPCs were identified in human hearts based on Sca-1 expression. These cells were isolated, and FACS, RT-PCR and immunocytochemistry were used to determine their baseline characteristics. Cardiomyogenic differentiation was induced by stimulation with 5-azacytidine.
Results
hCMPCs were localised within the atria, atrioventricular region, and epicardial layer of the foetal and adult human heart. In vitro, hCMPCs could be induced to differentiate into cardiomyocytes and formed spontaneously beating aggregates, without the need for co-culture with neonatal cardiomyocytes.
Conclusion
The human heart harbours a pool of resident cardiomyocyte progenitor cells, which can be expanded and differentiated in vitro. These cells may provide a suitable source for cardiac regeneration cell therapy. (Neth Heart J 2008;16:163-9.)
PMCID: PMC2431168  PMID: 18566670
human cardiac progenitor cell; cardiomyocytes; differentiation
17.  Sca-1+ Cardiosphere-Derived Cells Are Enriched for Isl1-Expressing Cardiac Precursors and Improve Cardiac Function after Myocardial Injury 
PLoS ONE  2012;7(1):e30329.
Background
Endogenous cardiac progenitor cells are a promising option for cell-therapy for myocardial infarction (MI). However, obtaining adequate numbers of cardiac progenitors after MI remains a challenge. Cardiospheres (CSs) have been proposed to have cardiac regenerative properties; however, their cellular composition and how they may be influenced by the tissue milieu remains unclear.
Methodology/Principal Finding
Using “middle aged” mice as CSs donors, we found that acute MI induced a dramatic increase in the number of CSs in a mouse model of MI, and this increase was attenuated back to baseline over time. We also observed that CSs from post-MI hearts engrafted in ischemic myocardium induced angiogenesis and restored cardiac function. To determine the role of Sca-1+CD45- cells within CSs, we cloned these from single cell isolates. Expression of Islet-1 (Isl1) in Sca-1+CD45- cells from CSs was 3-fold higher than in whole CSs. Cloned Sca-1+CD45- cells had the ability to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells in vitro. We also observed that cloned cells engrafted in ischemic myocardium induced angiogenesis, differentiated into endothelial and smooth muscle cells and improved cardiac function in post-MI hearts.
Conclusions/Significance
These studies demonstrate that cloned Sca-1+CD45- cells derived from CSs from infarcted “middle aged” hearts are enriched for second heart field (i.e., Isl-1+) precursors that give rise to both myocardial and vascular tissues, and may be an appropriate source of progenitor cells for autologous cell-therapy post-MI.
doi:10.1371/journal.pone.0030329
PMCID: PMC3260268  PMID: 22272337
18.  Functional Cardiomyocytes Derived from Isl1 Cardiac Progenitors via Bmp4 Stimulation 
PLoS ONE  2014;9(12):e110752.
As heart failure due to myocardial infarction remains a leading cause of morbidity worldwide, cell-based cardiac regenerative therapy using cardiac progenitor cells (CPCs) could provide a potential treatment for the repair of injured myocardium. As adult CPCs may have limitations regarding tissue accessibility and proliferative ability, CPCs derived from embryonic stem cells (ESCs) could serve as an unlimited source of cells with high proliferative ability. As one of the CPCs that can be derived from embryonic stem cells, Isl1 expressing cardiac progenitor cells (Isl1-CPCs) may serve as a valuable source of cells for cardiac repair due to their high cardiac differentiation potential and authentic cardiac origin. In order to generate an unlimited number of Isl1-CPCs, we used a previously established an ESC line that allows for isolation of Isl1-CPCs by green fluorescent protein (GFP) expression that is directed by the mef2c gene, specifically expressed in the Isl1 domain of the anterior heart field. To improve the efficiency of cardiac differentiation of Isl1-CPCs, we studied the role of Bmp4 in cardiogenesis of Isl1-CPCs. We show an inductive role of Bmp directly on cardiac progenitors and its enhancement on early cardiac differentiation of CPCs. Upon induction of Bmp4 to Isl1-CPCs during differentiation, the cTnT+ cardiomyocyte population was enhanced 2.8±0.4 fold for Bmp4 treated CPC cultures compared to that detected for vehicle treated cultures. Both Bmp4 treated and untreated cardiomyocytes exhibit proper electrophysiological and calcium signaling properties. In addition, we observed a significant increase in Tbx5 and Tbx20 expression in differentiation cultures treated with Bmp4 compared to the untreated control, suggesting a link between Bmp4 and Tbx genes which may contribute to the enhanced cardiac differentiation in Bmp4 treated cultures. Collectively these findings suggest a cardiomyogenic role for Bmp4 directly on a pure population of Isl1 expressing cardiac progenitors, which could lead to enhancement of cardiac differentiation and engraftment, holding a significant therapeutic value for cardiac repair in the future.
doi:10.1371/journal.pone.0110752
PMCID: PMC4270687  PMID: 25522363
19.  Embryonic stem cell-derived cardiomyocytes harbor a subpopulation of niche-forming Sca-1+ progenitor cells 
Molecular and Cellular Biochemistry  2010;349(1-2):69-76.
The adult mammalian heart is known to contain a population of cardiac progenitor cells. It has not been unambiguously determined, however, whether these cells form as part of the developmental program of the heart or migrate there by way of the circulatory system. This study was done in order to determine the origin of this population of cells. A population of cardiomyocytes was established from mouse embryonic stem (ES) cells using a genetic selection technique. In order to determine whether cardiac progenitor cells exist within this ES cell-derived cardiomyocyte population, the cells were analyzed by fluorescence activated cell sorting (FACS) using an antibody directed against stem cell antigen-1 (Sca-1). We observed that approximately 4% of the cardiomyocyte population was composed of Sca-1+ cells. When the Sca-1+ cells were isolated by magnetic cell sorting and differentiated as cellular aggregates, contractions were observed in 100% of the aggregates. Gene expression studies using quantitative RT-PCR showed that these cells expressed terminally differentiated cardiac-specific genes. When three-dimensional cellular aggregates were formed from ES cell-derived cardiomyocytes co-cultured with adult HL-1 cardiomyocytes, the Sca-1+ cells were found to “sort out” and form niches within the cell aggregates. Our data demonstrate that cardiac progenitor cells in the adult heart originate as part of the developmental program of the heart and that Sca-1+ progenitor cells can provide an important in vitro model system to study the formation of cellular niches in the heart.
doi:10.1007/s11010-010-0661-9
PMCID: PMC3394185  PMID: 21127947
Cardiomyocytes; Cardiac progenitor cells; Cardiac niche; Sca-1; Mouse embryonic stem cells; ES cells
20.  Contribution of Bone Marrow-Derived Hematopoietic Stem/Progenitor Cells to the Generation of Donor-Marker+ Cardiomyocytes In Vivo 
PLoS ONE  2013;8(5):e62506.
Background
Definite identification of the cell types and the mechanism relevant to cardiomyogenesis is essential for effective cardiac regenerative medicine. We aimed to identify the cell populations that can generate cardiomyocytes and to clarify whether generation of donor-marker+ cardiomyocytes requires cell fusion between BM-derived cells and recipient cardiomyocytes.
Methodology/Principal Findings
Purified BM stem/progenitor cells from green fluorescence protein (GFP) mice were transplanted into C57BL/6 mice or cyan fluorescence protein (CFP)-transgenic mice. Purified human hematopoietic stem cells (HSCs) from cord blood were transplanted into immune-compromised NOD/SCID/IL2rγnull mice. GFP+ cells in the cardiac tissue were analyzed for the antigenecity of a cardiomyocyte by confocal microscopy following immunofluorescence staining. GFP+ donor-derived cells, GFP+CFP+ fused cells, and CFP+ recipient-derived cells were distinguished by linear unmixing analysis. Hearts of xenogeneic recipients were evaluated for the expression of human cardiomyocyte genes by real-time quantitative polymerase chain reaction. In C57BL/6 recipients, Lin−/lowCD45+ hematopoietic cells generated greater number of GFP+ cardiomyocytes than Lin−/lowCD45− mesenchymal cells (37.0+/−23.9 vs 0.00+/−0.00 GFP+ cardiomyocytes per a recipient, P = 0.0095). The number of transplanted purified HSCs (Lin−/lowSca-1+ or Lin−Sca-1+c-Kit+ or CD34−Lin−Sca-1+c-Kit+) showed correlation to the number of GFP+ cardiomyocytes (P<0.05 in each cell fraction), and the incidence of GFP+ cardiomyocytes per injected cell dose was greatest in CD34−Lin−Sca-1+c-Kit+ recipients. Of the hematopoietic progenitors, total myeloid progenitors generated greater number of GFP+ cardiomyocytes than common lymphoid progenitors (12.8+/−10.7 vs 0.67+/−1.00 GFP+ cardiomyocytes per a recipient, P = 0.0021). In CFP recipients, all GFP+ cardiomyocytes examined coexpressed CFP. Human troponin C and myosin heavy chain 6 transcripts were detected in the cardiac tissue of some of the xenogeneic recipients.
Conclusions/Significance
Our results indicate that HSCs resulted in the generation of cardiomyocytes via myeloid intermediates by fusion-dependent mechanism. The use of myeloid derivatives as donor cells could potentially allow more effective cell-based therapy for cardiac repair.
doi:10.1371/journal.pone.0062506
PMCID: PMC3647070  PMID: 23667482
21.  Cardiac Stem Cell Secretome Protects Cardiomyocytes from Hypoxic Injury Partly via Monocyte Chemotactic Protein-1-Dependent Mechanism 
Cardiac stem cells (CSCs) were known to secrete diverse paracrine factors leading to functional improvement and beneficial left ventricular remodeling via activation of the endogenous pro-survival signaling pathway. However, little is known about the paracrine factors secreted by CSCs and their roles in cardiomyocyte survival during hypoxic condition mimicking the post-myocardial infarction environment. We established Sca-1+/CD31− human telomerase reverse transcriptase-immortalized CSCs (Sca-1+/CD31− CSCshTERT), evaluated their stem cell properties, and paracrine potential in cardiomyocyte survival during hypoxia-induced injury. Sca-1+/CD31− CSCshTERT sustained proliferation ability even after long-term culture exceeding 100 population doublings, and represented multi-differentiation potential into cardiomyogenic, endothelial, adipogenic, and osteogenic lineages. Dominant factors secreted from Sca-1+/CD31− CSCshTERT were EGF, TGF-β1, IGF-1, IGF-2, MCP-1, HGF R, and IL-6. Among these, MCP-1 was the most predominant factor in Sca-1+/CD31− CSCshTERT conditioned medium (CM). Sca-1+/CD31− CSCshTERT CM increased survival and reduced apoptosis of HL-1 cardiomyocytes during hypoxic injury. MCP-1 silencing in Sca-1+/CD31− CSCshTERT CM resulted in a significant reduction in cardiomyocyte apoptosis. We demonstrated that Sca-1+/CD31− CSCshTERT exhibited long-term proliferation capacity and multi-differentiation potential. Sca-1+/CD31− CSCshTERT CM protected cardiomyocytes from hypoxic injury partly via MCP-1-dependent mechanism. Thus, they are valuable sources for in vitro and in vivo studies in the cardiovascular field.
doi:10.3390/ijms17060800
PMCID: PMC4926334  PMID: 27231894
cardiac stem cells; immortalization; secretome; MCP-1; cardiomyocyte survival
22.  Identification and Characterization of a Novel Multipotent Sub-Population of Sca-1+ Cardiac Progenitor Cells for Myocardial Regeneration 
PLoS ONE  2011;6(9):e25265.
Methods and Results
The cardiac stem/progenitor cells from adult mice were seeded at low density in serum-free medium. The colonies thus obtained were expanded separately and assessed for expression of stem cell antigen-1 (Sca-1). Two colonies each with high Sca-1 (CSH1; 95.9%; CSH2; 90.6%) and low Sca-1 (CSL1; 37.1%; CSL2; 17.4%) expressing cells were selected for further studies. Sca-1+ cells (98.4%) isolated using Magnetic Cell Sorting System (MACS) from the hearts were used as a control. Although the selected populations were similar in surface marker expression (low in c-kit, CD45, CD34, CD31 and high in CD29), these cells exhibited diverse differentiation potential. Unlike CSH1, CSH2 expressed Nanog, TERT, Bcrp1, Nestin, Musashi1 and Isl-1, and also showed differentiation into osteogenic, chondrogenic, smooth muscle, endothelial and cardiac lineages. MACS sorted cells exhibited similar tendency albeit with relatively weaker differentiation potential. Transplantation of CSH2 cells into infarcted heart showed attenuated infarction size, significantly preserved left ventricular function and anterior wall thickness, and increased capillary density. We also observed direct differentiation of transplanted cells into endothelium and cardiomyocytes.
Conclusions
The cardiac stem/progenitor cells isolated by a combined clonal selection and surface marker approach possessed multiple stem cell features important for cardiac regeneration.
doi:10.1371/journal.pone.0025265
PMCID: PMC3182214  PMID: 21980409
23.  Characterization of contracting cardiomyocyte colonies in the primary culture of neonatal rat myocardial cells 
Cell Cycle  2014;13(6):910-918.
The unmet clinical need for myocardial repair after irreversible ischemic injury requires a better understanding of the biological properties of cardiac stem cells (CSCs). Using a primary culture of neonatal rat myocardial cells, we describe the formation and maturation of contracting cardiomyocyte colonies stemming from c-kit+, Sca+, or Isl1+ CSCs, which occurs in parallel to the hypertrophy of the major cardiac myocyte population. The contracting cardiomyocyte colonies (~1–2 colonies per 1 × 105 of myocardial cells) were identified starting from eighth day of culturing. At first, spontaneous weak, asynchronous, and arrhythmic contractions of the colonies at a rate of 2–3 beats/min were registered. Over time, the contractions of the colonies became more synchronous and frequent, with a contraction rate of 58–60 beats/min by the 30th day of culturing. The colonies were characterized by the CSCs subtype-specific pattern of growth and structure. The cells of the colonies were capable of spontaneous cardiomyogenic differentiation, demonstrating expression of both sarcomeric α-actinin and α-sarcomeric actin as well as the maturation of contractile machinery and typical Ca2+ responses to caffeine (5 mМ) and K+ (120 mМ). Electromechanical coupling, characterized by cardiac muscle-specific Ca2+-induced Ca2+ release, was evident at 3 weeks of culturing. Thus, the co-cultivation of CSCs with mature cardiac cells resulted in the formation of contracting cardiomyocyte colonies, resembling the characteristics of in vivo cardiomyogenesis. The proposed model can be used for the investigation of fundamental mechanisms underlying cardiomyogenic differentiation of CSCs as well as for drug testing and/or other applications.
doi:10.4161/cc.27768
PMCID: PMC3984314  PMID: 24423725
primary culture of neonatal myocardial cells; cardiac myocytes; resident cardiac stem cells; proliferation; differentiation; contracting cardiomyocyte colonies; electromechanical coupling; Ca2+-induced Ca2+ release
24.  Isolation and characterization of a Sca-1+/CD31- progenitor cell lineage derived from mouse heart tissue 
BMC Biotechnology  2014;14:75.
Background
Myocardial infarction remains the leading cause of mortality in developed countries despite recent advances in its prevention and treatment. Regenerative therapies based on resident cardiac progenitor cells (CPCs) are a promising alternative to conventional treatments. However, CPCs resident in the heart are quite rare. It is unclear how these CPCs can be isolated and cultured efficiently and what the effects of long-term culture in vitro are on their ‘stemness’ and differentiation potential, but this is critical knowledge for CPCs’ clinical application.
Results
Here, we isolated stem cell antigen-1 positive cells from postnatal mouse heart by magnetic active cell sorting using an iron-labeled anti-mouse Sca-1 antibody, and cultured them long-term in vitro. We tested stemness marker expression and the proliferation ability of long-term cultured Sca-1+ cells at early, middle and late passages. Furthermore, we determined the differentiation potential of these three passages into cardiac cell lineages (cardiomyocytes, smooth muscle and endothelial cells) after induction in vitro. The expression of myocardial, smooth muscle and endothelial cell-specific genes and surface markers were analyzed by RT-PCR and IF staining. We also investigated the oncogenicity of the three passages by subcutaneously injecting cells in nude mice. Overall, heart-derived Sca-1+ cells showed CPC characteristics: long-term propagation ability in vitro, non-tumorigenic in vivo, persistent expression of stemness and cardiac-specific markers, and multipotent differentiation into cardiac cell lineages.
Conclusions
Our research may bring new insights to myocardium regeneration, for which even a small number of biopsy-derived CPCs could be enriched and propagated long term in vitro to obtain sufficient seed cells for cell injection or cardiac tissue engineering.
doi:10.1186/1472-6750-14-75
PMCID: PMC4133720  PMID: 25106452
Cardiac progenitor cell; Stem cell antigen-1; Differentiation; Multipotent; Self-renewal
25.  Bone Marrow-Derived Human Mesenchymal Stem Cells Express Cardiomyogenic Proteins But Do Not Exhibit Functional Cardiomyogenic Differentiation Potential 
Stem Cells and Development  2012;21(13):2457-2470.
Despite their paracrine activites, cardiomyogenic differentiation of bone marrow (BM)-derived mesenchymal stem cells (MSCs) is thought to contribute to cardiac regeneration. To systematically evaluate the role of differentiation in MSC-mediated cardiac regeneration, the cardiomyogenic differentiation potential of human MSCs (hMSCs) and murine MSCs (mMSCs) was investigated in vitro and in vivo by inducing cardiomyogenic and noncardiomyogenic differentiation. Untreated hMSCs showed upregulation of cardiac tropopin I, cardiac actin, and myosin light chain mRNA and protein, and treatment of hMSCs with various cardiomyogenic differentiation media led to an enhanced expression of cardiomyogenic genes and proteins; however, no functional cardiomyogenic differentiation of hMSCs was observed. Moreover, co-culturing of hMSCs with cardiomyocytes derived from murine pluripotent cells (mcP19) or with murine fetal cardiomyocytes (mfCMCs) did not result in functional cardiomyogenic differentiation of hMSCs. Despite direct contact to beating mfCMCs, hMSCs could be effectively differentiated into cells of only the adipogenic and osteogenic lineage. After intramyocardial transplantation into a mouse model of myocardial infarction, Sca-1+ mMSCs migrated to the infarcted area and survived at least 14 days but showed inconsistent evidence of functional cardiomyogenic differentiation. Neither in vitro treatment nor intramyocardial transplantation of MSCs reliably generated MSC-derived cardiomyocytes, indicating that functional cardiomyogenic differentiation of BM-derived MSCs is a rare event and, therefore, may not be the main contributor to cardiac regeneration.
doi:10.1089/scd.2011.0626
PMCID: PMC3425038  PMID: 22309203

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