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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.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  Immunogenicity when utilizing adenovirus serotype 4 and 5 vaccines expressing circumsporozoite protein in naïve and Adenovirus (Ad5) immune mice 
Malaria Journal  2012;11:209.
Background
Induction of potent long lasting effector T cell responses against liver stage malaria antigens strongly correlates with protection from malaria. While Adenovirus serotype 5 (Ad5) based malaria vaccine platforms have the ability to induce potent effector T cell responses against transgenes, high rates of pre-existing Ad5 immunity in malaria endemic regions has prompted study of alternative Ad serotype based malaria vaccines as replacements for Ad5 based malaria vaccines. The research described in this article examines the utility of alternative serotype adenovirus serotype 4 (Ad4) expressing a sporozoite surface protein (circumsporozoite protein (CSP)) (Ad4-CSP) to induce immune responses against CSP. The immunogenicity of Ad4-CSP was also tested in homologous and heterologous prime boost vaccinations in both Ad5 naïve and Ad5 immune backgrounds as compared to use of Ad5-CSP.
Results
In Ad5 naïve animals, use of Ad4-CSP priming vaccinations followed by boosting with Ad5-CSP (Ad4-CSP/Ad5-CSP) maximally increased the numbers of CSP specific cytokine secreting cytotoxic T cells relative to repeated use of Ad5-CSP. The Ad4-CSP/Ad5-CSP regimen also induced equivalent levels of CSP specific cell killing as did homologous prime-boost vaccinations with Ad5-CSP, despite stimulating lower numbers of CSP specific cytotoxic T cells. Priming with Ad4-CSP followed by a homologous boost resulted in significantly less CSP specific humoral responses than any other vaccination regimen tested in Ad naïve animals. In Ad5 immune animals, addition of Ad4-CSP in homologous or heterologous prime boost resulted in inductions of higher CSP specific responses than animals repeatedly vaccinated with Ad5-CSP alone. However, the observed responses were well below those observed in similarly treated Ad naïve mice.
Conclusions
While the Ad4-CSP/Ad5-CSP and Ad5-CSP/Ad5-CSP vaccination regimens resulted in equivalent CSP specific killing in Ad naïve animals, Ad4-CSP/Ad5-CSP achieved this result with a lower percentage of CSP specific CD8+ T cells and a higher number of IFNγ secreting cells, suggesting that the Ad4-CSP/Ad5-CSP vaccination regimen elicits more efficient cytotoxic T cells. In Ad5 immune animals use of Ad4-CSP improved CSP specific immune responses as compared to repeated use of Ad5-CSP, but could not achieve the levels of immunogenicity observed when the same vaccine regimens were used in Ad naïve animals. These data indicate the existence of some level of immunological cross-reactivity between these two adenovirus subgroups. Based on these results, it is suggested that future studies should undertake similarly stringent analyses of alternative Ad serotypes to establish their effectiveness as replacements for Ad5.
doi:10.1186/1475-2875-11-209
PMCID: PMC3472263  PMID: 22720732
Serotype 5; Serotype 4; Adenovirus; Malaria; Circumsporozoite protein; Vaccine; Heterologous; Homologous; Prime; Boost
13.  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
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.  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
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.  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
18.  Relative Roles of Direct Regeneration Versus Paracrine Effects of Human Cardiosphere-Derived Cells Transplanted Into Infarcted Mice 
Circulation research  2010;106(5):971-980.
Rationale
Multiple biological mechanisms contribute to the efficacy of cardiac cell therapy. Most prominent among these are direct heart muscle and blood vessel regeneration from transplanted cells, as opposed to paracrine enhancement of tissue preservation and/or recruitment of endogenous repair.
Objective
Human cardiac progenitor cells, cultured as cardiospheres (CSps) or as CSp-derived cells (CDCs), have been shown to be capable of direct cardiac regeneration in vivo. Here we characterized paracrine effects in CDC transplantation and investigated their relative importance versus direct differentiation of surviving transplanted cells.
Methods and Results
In vitro, many growth factors were found in media conditioned by human adult CSps and CDCs; CDC-conditioned media exerted antiapoptotic effects on neonatal rat ventricular myocytes, and proangiogenic effects on human umbilical vein endothelial cells. In vivo, human CDCs secreted vascular endothelial growth factor, hepatocyte growth factor, and insulin-like growth factor 1 when transplanted into the same SCID mouse model of acute myocardial infarction where they were previously shown to improve function and to produce tissue regeneration. Injection of CDCs in the peri-infarct zone increased the expression of Akt, decreased apoptotic rate and caspase 3 level, and increased capillary density, indicating overall higher tissue resilience. Based on the number of human-specific cells relative to overall increases in capillary density and myocardial viability, direct differentiation quantitatively accounted for 20% to 50% of the observed effects.
Conclusions
Together with their spontaneous commitment to cardiac and angiogenic differentiation, transplanted CDCs serve as “role models,” recruiting endogenous regeneration and improving tissue resistance to ischemic stress. The contribution of the role model effect rivals or exceeds that of direct regeneration.
doi:10.1161/CIRCRESAHA.109.210682
PMCID: PMC4317351  PMID: 20110532
paracrine hypothesis; cardiac stem cells; VEGF; HGF; IGF1
19.  Reduced Expression of Inflammatory Genes in Deceased Donor Kidneys Undergoing Pulsatile Pump Preservation 
PLoS ONE  2012;7(4):e35526.
Background
The use of expanded criteria donor kidneys (ECD) had been associated with worse outcomes. Whole gene expression of pre-implantation allograft biopsies from deceased donor kidneys (DDKs) was evaluated to compare the effect of pulsatile pump preservation (PPP) vs. cold storage preservation (CSP) on standard and ECD kidneys.
Methodology/Principal Findings
99 pre-implantation DDK biopsies were studied using gene expression with GeneChips. Kidneys transplant recipients were followed post transplantation for 35.8 months (range = 24–62). The PPP group included 60 biopsies (cold ischemia time (CIT)  = 1,367+/−509 minutes) and the CSP group included 39 biopsies (CIT = 1,022+/−485 minutes) (P<0.001). Donor age (42.0±14.6 vs. 34.1±14.2 years, P = 0.009) and the percentage of ECD kidneys (PPP = 35% vs. CSP = 12.8%, P = 0.012) were significantly different between groups. A two-sample t-test was performed, and probe sets having a P<0.001 were considered significant. Probe set level linear models were fit using cold ischemia time and CSP/PPP as independent variables to determine significant probe sets (P<0.001) between groups after adjusting for cold ischemia time. Thus, 43 significant genes were identified (P<0.001). Over-expression of genes associated with inflammation (CD86, CD209, CLEC4, EGFR2, TFF3, among others) was observed in the CSP group. Cell-to-cell signaling and interaction, and antigen presentation were the most important pathways with genes significantly over-expressed in CSP kidneys. When the analysis was restricted to ECD kidneys, genes involved in inflammation were also differentially up-regulated in ECD kidneys undergoing CSP. However, graft survival at the end of the study was similar between groups (P = 0.2). Moreover, the incidence of delayed graft function was not significant between groups.
Conclusions/Significance
Inflammation was the most important up-regulated pattern associated with pre-implantation biopsies undergoing CSP even when the PPP group has a larger number of ECD kidneys. No significant difference was observed in delayed graft function incidence and graft function post-transplantation. These findings support the use of PPP in ECD donor kidneys.
doi:10.1371/journal.pone.0035526
PMCID: PMC3335841  PMID: 22545113
20.  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
21.  Murine Cardiosphere-Derived Cells Are Impaired by Age but Not by Cardiac Dystrophic Dysfunction 
Stem Cells and Development  2013;23(9):1027-1036.
To be clinically relevant as a therapy for heart failure, endogenous progenitor cells must be isolated and expanded from aged and/or diseased tissue. Here, we investigated the effect of age and cardiac impairment resulting from lack of dystrophin on murine cardiosphere-derived cells (CDCs). CDCs were isolated and expanded from atrial biopsies from wild-type mice aged 1.5, 6, 18, and 24 months and from mdx mice aged 6 and 18 months. Cardiac function was measured in mdx mice and age-matched wild-type mice using high-resolution cine magnetic resonance imaging. CDCs could be isolated and expanded from all mice, however, the number of cells obtained, and their regenerative potential, decreased with age, as demonstrated by decreased expression of stem cell markers, c-kit and Sca-1, and decreased cell proliferation, migration, clonogenicity, and differentiation. Six-month-old mdx mice showed right ventricular (RV) dilation and reduced RV ejection fraction (EF) in comparison to wild-type mice. Older mdx mice displayed significant RV and left ventricular dilation and decreased EF in both ventricles, compared with age-matched wild-type mice. Mdx mouse hearts contained significantly more fibrotic tissue than age-matched wild-type mouse hearts. However, CDCs isolated from mice aged 6 and 18 months had the same number and regenerative potential from mdx mice and age-matched wild-type mice. Thus, the cardiac progenitor cell population is impaired by age but is not substantially altered by the progressive deterioration in function of the dystrophic heart.
doi:10.1089/scd.2013.0388
PMCID: PMC3996995  PMID: 24351030
22.  Characterization of lung stem cell niches in a mouse model of bleomycin-induced fibrosis 
Introduction
In lung fibrosis, alveolar epithelium degenerates progressively. The goal of regenerative medicine is to aid repair and regeneration of the lost tissues in parenchyma and airways for which mobilization of tissue-resident endogenous or bone marrow-derived exogenous stem cells niches is a critical step. We used a lung injury model in mice to identify and characterize functional lung stem cells to clarify how stem cell niches counteract this degenerative process.
Methods
Short term assay (STA) - Bleomycin-induced lung inflammation and fibrosis were assessed in a model of idiopathic pulmonary fibrosis in wild-type (WT), gp91phox-/- (NOX-/-), and gp91phoxMMP-12 double knockout (DKO) mice on C57Bl/6 background and Hoechst 33322 dye effluxing side population (SP) cells characterized. Long term assay (LTA) - In a bleomycin induced lung fibrosis model in C57Bl6 mice, the number of mature cells were quantified over 7, 14, and 21 days in bone marrow (BM), peripheral blood (PB), lung parenchyma (LP) and brochoalveolar lavage (BAL) fluid by FACS. BrdU pulse chase experiment (10 weeks) was used to identify label retaining cells (LRC). BrdU+ and BrdU- cells were characterized by hematopoietic (CD45+), pluripotency (TTF1+, Oct3/4+, SSEA-3+, SSEA-4+, Sca1+, Lin-, CD34+, CD31+), and lung lineage-specific (SPC+, AQP-5+, CC-10+) markers. Clonogenic potential of LRCs were measured by CFU-c assays.
Results
STA- In lung, cellularity increased by 5-fold in WT and 6-fold in NOX-/- by d7. Lung epithelial markers were very low in expression in all SP flow sorted from lung of all three genotypes cultured ex vivo. (p < 0.01). Post-bleomycin, the SP in NOX-/- lung increased by 3.6-fold over WT where it increased by 20-fold over controls. Type I and II alveolar epithelial cells progressively diminished in all three genotypes by d21 post-bleomycin. D7 post-bleomycin, CD45+ cells in BALf in NOX-/- was 1.7-fold > WT, 57% of which were Mf that decreased by 67% in WT and 83% in NOX-/- by d21.LTA- Cellularity as a factor of time remained unchanged in BM, PB, LP and BAL fluid. BrdU+ (LRC) were the putative stem cells. BrdU+CD45+ cells increased by 0.7-fold and SPC+CC10+ bronchoalveolar stem cells (BASC), decreased by ~40-fold post-bleomycin. BrdU+VEGF+ cells decreased by 1.8-fold while BrdU-VEGF+ cells increased 4.6-fold. Most BrdU- cells were CD45-. BrdU- BASCs remained unchanged post-bleomycin. CFU-c of the flow-sorted BrdU+ cells remained similar in control and bleomycin-treated lungs.
Conclusion
STA- Inflammation is a pre-requisite for fibrosis; SP cells, being the putative stem cells in the lungs, were increased (either by self renewal or by recruitment from the exogenous bone marrow pool) post-bleomycin in NOX-/- but not in DKO indicating the necessity of cross-talk between gp91phox and MMP-12 in this process; ex vivo cultured SP progressively lose pluripotent markers, notably BASC (SPC+CC10+) - significance is unknown. LTA- The increase in the hematopoietic progenitor pool in lung indicated that exogenous progenitors from circulation contribute to lung regeneration. Most non-stem cells were non-hematopoietic in origin indicating that despite tissue turnover, BASCs are drastically depleted possibly necessitating recruitment of progenitors from the hematopoietic pool. Loss of VEGF+ LRC may indicate a signal for progenitor mobilization from niches. BrdU- BASC population may be a small quiescent population that remains as a reserve for more severe lung injury. Increase in VEGF+ non-LRC may indicate a checkpoint to counterbalance the mobilization of VEGF+ cells from the stem cell niche.
doi:10.1186/scrt112
PMCID: PMC3392768  PMID: 22643035
23.  A Hydrophobic Patch in the Competence-Stimulating Peptide, a Pneumococcal Competence Pheromone, Is Essential for Specificity and Biological Activity 
Journal of Bacteriology  2006;188(5):1744-1749.
Induction of competence for natural genetic transformation in Streptococcus pneumoniae depends on pheromone-mediated cell-cell communication and a signaling pathway consisting of the competence-stimulating peptide (CSP), its membrane-embedded histidine kinase receptor ComD, and the cognate response regulator ComE. Extensive screening of pneumococcal isolates has revealed that two major CSP variants, CSP1 and CSP2, are found in members of this species. Even though the primary structures of CSP1 and CSP2 are about 50% identical, they are highly specific for their respective receptors, ComD1 and ComD2. In the present work, we have investigated the structural basis of this specificity by determining the three-dimensional structure of CSP1 from nuclear magnetic resonance data and comparing the agonist activity of a number of CSP1/CSP2 hybrid peptides toward the ComD1 and ComD2 receptors. Our results show that upon exposure to membrane-mimicking environments, the 17-amino-acid CSP1 pheromone adopts an amphiphilic α-helical configuration stretching from residue 6 to residue 12. Furthermore, the pattern of agonist activity displayed by the various hybrid peptides revealed that hydrophobic amino acids, some of which are situated on the nonpolar side of the α-helix, strongly contribute to CSP specificity. Together, these data indicate that the identified α-helix is an important structural feature of CSP1 which is essential for effective receptor recognition under natural conditions.
doi:10.1128/JB.188.5.1744-1749.2006
PMCID: PMC1426553  PMID: 16484185
24.  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
25.  Sca-1 Knockout Impairs Myocardial and Cardiac Progenitor Cell Function 
Circulation research  2012;111(6):750-760.
Rationale
Cardiac progenitor cells are important for maintenance of myocardial structure and function, but molecular mechanisms governing these progenitor cells remain obscure and require elucidation to enhance regenerative therapeutic approaches.
Objective
To understand consequences of stem cell antigen-1 (Sca-1) deletion upon functional properties of c-kit+ cardiac progenitor cells and myocardial performance using a Sca-1 knockout/Green Fluorescent Protein knock-in reporter mouse (ScaKI).
Methods and Results
Genetic deletion of Sca-1 results in early-onset cardiac contractile deficiency as determined by echocardiography and hemodynamics as well as age-associated hypertrophy. Resident cardiac progenitor cells in ScaKI mice do not respond to pathological damage in vivo, consistent with observations of impaired growth and survival of ScaKI cardiac progenitor cells in vitro. The molecular basis of the defect in ScaKI cardiac progenitor cells is associated with increased canonical Wnt signaling pathway activation consistent with molecular characteristics of lineage commitment.
Conclusions
Genetic deletion of Sca-1 causes primary cardiac defects in myocardial contractility and repair consistent with impairment of resident cardiac progenitor cell proliferative capacity associated with altered canonical Wnt signaling.
doi:10.1161/CIRCRESAHA.112.274662
PMCID: PMC3463406  PMID: 22800687
Sca-1; c-kit; heart; cardiac progenitor cell; infarction; myocardium; Sca-1 knock-out; β–catenin; cardiac development

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