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.
Adult stem cells; Cardiac progenitor cells; Cardiogenesis; Cardiovascular disease; Regeneration
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.
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.
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.
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.
cardiac side population cells; Wnt signaling; cardiac regeneration; stem cells; proliferation
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.
Abcg2; Cardiomyocytes; Cell death; Contractility; Differentiation; Mdr1; Progenitor cells; Proliferation; SP cells; Stem cells
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.
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.
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
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
human cardiac progenitor cell; cardiomyocytes; differentiation
Because heat shock proteins have been shown to play a critical role in protecting cells from hyperthermia and other types of physiological stresses, it was of interest to determine what effect age and caloric restriction have on the ability of cells to regulate the expression of heat shock protein 70 (hsp70), the most prominent and most evolutionarily conserved of the heat shock proteins. Caloric restriction is the only experimental manipulation known to retard aging and increase survival of mammals. The ability of hepatocytes isolated from young/adult (4- to 7-month-old) and old (22- to 28-month-old) male Fischer F344 rats fed ad libitum or a caloric restriction diet (60% of the content of the ad libitum diet) to express hsp70 was determined after a mild heat shock (42.5 degrees C for 30 min). We found that the induction of hsp70 synthesis and mRNA levels by heat shock was 40 to 50% lower in hepatocytes isolated from old rats than in hepatocytes isolated from young rats. Using in situ hybridization, we found that essentially all hepatocytes from the young/adult and old rats expressed hsp70 in response to heat shock; therefore, the age-related decrease in the induction of hsp70 expression was not due to an age-related accumulation of cells that do not respond to heat shock. Measurements of hsp70 mRNA stability and hsp70 transcription demonstrated that the age-related decline in hsp70 expression arose from a decline in hsp70 transcription. Interestingly, the age-related decline in the induction of hsp70 expression was reversed by caloric restriction; e.g., the induction of hsp70 synthesis, mRNA levels, and nuclear transcription were significantly higher in hepatocytes isolated from old rats fed the caloric restricted diet than in hepatocytes isolated from old rats fed ad libitum. The levels of the heat shock transcription factor in nuclear extracts isolated from heat-shocked hepatocytes were measured in a gel shift assay. Binding of the heat shock transcription factor to the heat shock element decreased with age and was significantly higher in hepatocyte extracts isolated from old rats fed the caloric restriction diet than in those from old rats fed ad libitum. Thus, our study demonstrates that the ability of hepatocytes to respond to hyperthermia and express hsp70 decreases significantly with age and that this decrease occurs at the transcriptional level. In addition, caloric restriction, which retards aging, reversed the age-related decline in the induction of hsp70 transcription in hepatocytes.
Loss of cardiac mitochondrial function with age may cause increased cardiomyocyte death through mitochondria-mediated release of apoptogenic factors. We investigated ventricular subsarcolemmal (SSM) and interfibrillar (IFM) mitochondrial bioenergetics and susceptibility towards Ca2+-induced permeability transition pore (mPTP) opening with aging and lifelong calorie restriction (CR). Cardiac mitochondria were isolated from 8, 18, 29 and 37-month-old male Fischer 344 × Brown Norway rats fed either ad libitum (AL) or 40% calorie restricted diets. With age, H2O2 generation did not increase and oxygen consumption did not significantly decrease in either SSM or IFM. Strikingly, IFM displayed an increased susceptibility towards mPTP opening during senescence. In contrast, Ca2+ retention capacity of SSM was not affected by age, but SSM tolerated much less Ca2+ than IFM. Only modest age-dependent increases in cytosolic caspase activities and cytochrome c levels were observed and were not affected by CR. Levels of putative mPTP-modulating components: cyclophilin-D, the adenine nucleotide translocase (ANT), and the voltage-dependent ion channel (VDAC) were not affected by aging or CR. In summary, the age-related reduction of Ca2+ retention capacity in IFM may explain the increased susceptibility to stress-induced cell death in the aged myocardium.
heart disease; ischemia-reperfusion; senescence; hypertrophy
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.
Cardiomyocytes; Cardiac progenitor cells; Cardiac niche; Sca-1; Mouse embryonic stem cells; ES cells
Bone mass is correlated with body weight during growth. However, it is unclear how bone mass is influenced by weight gain following skeletal maturity. The purpose of this study was to determine the effects of weight maintenance and two rates of weight gain on bone metabolism using skeletally mature female rats. Eight-month-old female rats were fed one of 3 diets for 13 weeks: Lieber DeCarli liquid diet ad libitum (control diet), the same diet with caloric restriction to maintain initial body weight (calorie-restricted diet), and the same diet fed ad lib with the exception that appetite was enhanced (calorie-increased diet) by replacing a small quantity of maltose-dextran isolcalorically with ethanol (0.5% caloric intake). Compared to baseline, rats fed the calorie-restricted, control, and calorie-increased diets changed in weight by −1 ± 2% (mean ± SE), 10 ± 3%, and 21 ± 2%, respectively. Weight gain was associated with a significant increase in serum leptin, a putative regulator of bone formation. In contrast, significant differences in tibial bone mineral content and density were not detected among treatments groups following dietary intervention or between treatment groups and the baseline group. Similarly, indices of cancellous bone architecture (area, trabecular number, thickness, and separation) and bone turnover (mineralizing perimeter, mineral apposition rate, and bone formation rate) did not differ among groups following dietary intervention. Our findings suggest that neither weight gain nor increased serum leptin levels, over the range evaluated, influence bone metabolism in skeletally mature female rats.
Bone turnover; caloric restriction; osteoporosis; obesity; leptin
Cell based therapies hold promise of repairing an injured heart, and the description of stem and progenitor cells with cardiomyogenic potential is critical to its realization. At the vanguard of these efforts are analyses of embryonic stem cells, which clearly have the capacity to generate large numbers of cardiomyocytes in vitro. Through the use of this model system, a number of signaling mechanisms have been worked out that describes at least partially the process of cardiopoiesis. Studies on adult stem and on progenitor cells with cardiomyogenic potential are still in their infancy, and much less is known about the molecular signals that are required to induce the differentiation to cardiomyocytes. It is also unclear whether the pathways are similar or different between embryonic and adult cell-induced cardiomyogenesis, partly because of the continued controversies that surround the stem cell theory of cardiac self-renewal. Irrespective of any perceived or actual limitations, the study of stem and progenitor cells has provided important insights into the process of cardiomyogenesis, and it is likely that future research in this area will turn the promise of repairing an injured heart into a reality.
Embryonic Stem Cells; Adult Stem Cells; Cardiomyocytes; Cardiomyogenesis; Plasticity
While Bone-marrow endothelial progenitor cell based therapies (BM-EPC) improve the symptoms in patients with ischemic heart disease their limited plasticity and decreased function in patients with existing heart disease limits the full benefit of EPC therapy for cardiac regenerative medicine.
We hypothesized that reprogramming mouse and/or human EPCs using small molecules targeting key epigenetic repressive marks would lead to a global increase in active gene transcription, induce their cardiomyogenic potential and enhance their inherent angiogenic potential.
Method and Results
Mouse Lin-Sca1+CD31+ EPCs and human CD34+ cells were treated with inhibitors of DNA methyltransferases (5-Azacytidine), histone deacetylases (valproic acid) and G9a histone di-methyltransferase. Forty eight hour treatment led to global increase in active transcriptome including the reactivation of pluripotency associated and CMC specific mRNA expression while EC specific genes were significantly up-regulated. When cultured under appropriate differentiation conditions, reprogrammed EPCs showed efficient differentiation into CMC and vascular smooth muscle cells. Treatment with epigenetic modifying agents show marked increase in histone acetylation on cardiomyocyte and pluripotent cell specific gene promoters. Intra-myocardial transplantation of reprogrammed mouse and human EPCs in an acute myocardial infarction mouse model showed significant improvement in ventricular functions, which was histologically supported by their de novo CMC differentiation and increased capillary density and reduced fibrosis. Importantly, cell transplantation was safe and did not form teratomas.
Taken together, our results suggest that epigenetically reprogrammed EPCs display a safe, more plastic phenotype and improve post-infarct cardiac repair by both neo-cardiomyogenesis and neovascularization.
Epigenetic modification; EPC; cardiomyogenesis; myocardial ischemia; cell therapy; histone acetylation; trans-differentiation
Human studies indicate augmented myocardial lipid metabolism in females, and that sex and obesity interact to predict myocardial fatty acid oxidation and storage. Altered lipid dynamics precede cardiomyopathies, and many studies now address high fat diets. Conversely, caloric restriction (CR), is the most studied model for longevity and stress resistance, including protection against myocardial ischemia. However, no information exists on the effects of long-term caloric restriction (CR) on triacylglyceride (TAG) content and dynamics in the heart. This study explored the effects of CR, sex and age on TAG dynamics in mouse hearts. Male and female SVJ129 mice were fed either normal (ND) or CR diet for 3 or 10 months. In 5-month-old mice, CR similarly decreased cardiac TAG in males (ND: 25.5 +/− 4.5 nanomoles/mg protein; CR: 12.6 +/− 2.7, P<0.05) and females (ND: 30.1 +/− 4.4; CR: 13.7 +/− 1.2) (no significant differences in TAG content were seen between sexes). CR reduced the contribution of exogenous palmitate to oxidative metabolism in males and females, by 15% and 11% respectively, versus ND, without affecting cardiac workload. CR also induced a larger reduction in TAG turnover in male (68%) than female hearts (38%). Interestingly, in 5 month old male mice, CR reproduced the lower TAG turnover rates of middle-aged males (ND 13-month-old male = 423 +/−76 nanomoles/mg protein/min). Thus, long term CR reduces TAG pool dynamics. Despite reduced content, hearts of female mice subjected to CR retained a more dynamic TAG pool than males, while males respond with greater metabolic remodeling of cardiac lipid dynamics.
long term caloric restriction; triacylglyceride; long-chain fatty acids
Human mesenchymal stem cells (hMSCs) are broadly discussed as a promising cell population amongst others for regenerative therapy of ischemic heart disease and its consequences. Although cardiac-specific differentiation of hMSCs was reported in several in vitro studies, these results were sometimes controversial and not reproducible.
In our study we have analyzed different published protocols of cardiac differentiation of hMSCs and their modifications, including the use of differentiation cocktails, different biomaterial scaffolds, co-culture techniques, and two- and three-dimensional cultures. We also studied whether 5'-azacytidin and trichostatin A treatments in combination with the techniques mentioned above can increase the cardiomyogenic potential of hMSCs. We found that hMSCs failed to generate functionally active cardiomyocytes in vitro, although part of the cells demonstrated increased levels of cardiac-specific gene expression when treated with differentiation factors, chemical substances, or co-cultured with native cardiomyocytes.
The failure of hMSCs to form cardiomyocytes makes doubtful the possibility of their use for mechanical reparation of the heart muscle.
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.
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.
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.
Cellular senescence implies loss of proliferative and tissue regenerative capability. Also hypoxia, producing Reactive Oxygen Species (ROS), can damage cellular components through the oxidation of DNA, proteins and lipids, thus influencing the shortening of telomeres.
Since ribonucleoprotein Telomerase (TERT), catalyzing the replication of the ends of eukaryotic chromosomes, promotes cardiac muscle cell proliferation, hypertrophy and survival, here we investigated its role in the events regulating apoptosis occurrence and life span in hearts deriving from young and old rats exposed to hypoxia.
TUNEL (terminal-deoxinucleotidyl -transferase- mediated dUTP nick end-labeling) analysis reveals an increased apoptotic cell number in both samples after hypoxia exposure, mainly in the young with respect to the old. TERT expression lowers either in the hypoxic young, either in the old in both experimental conditions, with respect to the normoxic young. These events are paralleled by p53 and HIF-1 α expression dramatic increase and by p53/ HIF-1 α co-immunoprecipitation in the hypoxic young, evidencing the young subject as the most stressed by such challenge. These effects could be explained by induction of damage to genomic DNA by ROS that accelerates cell senescence through p53 activation. Moreover, by preventing TERT enzyme down-regulation, cell cycle exit and apoptosis occurrence could be delayed and new possibilities for intervention against cell ageing and hypoxia could be opened.
p53; telomerase; hypoxia; ageing; rat heart.
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.
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.
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.
Sca-1; c-kit; heart; cardiac progenitor cell; infarction; myocardium; Sca-1 knock-out; β–catenin; cardiac development
Cysteine-string protein (CSP) is an extensively palmitoylated DnaJ-family chaperone, which exerts an important neuroprotective function. Palmitoylation is required for the intracellular sorting and function of CSP, and thus it is important to understand how this essential modification of CSP is regulated. Recent work identified 23 putative palmitoyl transferases containing a conserved DHHC domain in mammalian cells, and here we show that palmitoylation of CSP is enhanced specifically by co-expression of the Golgi-localised palmitoyl transferases DHHC3, DHHC7, DHHC15 or DHHC17. Indeed, these DHHC proteins promote stable membrane attachment of CSP, which is otherwise cytosolic. An inverse correlation was identified between membrane affinity of unpalmitoylated CSP mutants and subsequent palmitoylation: mutants with an increased membrane affinity localise to the ER and are physically separated from the Golgi-localised DHHC proteins. Palmitoylation of an ER-localised mutant could be rescued by brefeldin A (BFA) treatment, which promotes the mixing of ER and Golgi membranes. Interestingly though, the palmitoylated mutant remained at the ER following BFA washout, and did not traffic to more distal membrane compartments. We propose that CSP has a weak membrane affinity that allows the protein to locate its partner Golgi-localised DHHC proteins directly by membrane ‘sampling’. Mutations that enhance membrane association prevent sampling and lead to accumulation of CSP on cellular membranes such as the ER. The coupling of CSP palmitoylation to Golgi membranes may thus be an important requirement for subsequent sorting.
cspA (for cell surface protein A) encodes a repeat-rich glycophosphatidylinositol (GPI)-anchored cell wall protein (CWP) in the pathogenic fungus Aspergillus fumigatus. The number of repeats in cspA varies among isolates, and this trait is used for typing closely related strains of A. fumigatus. We have previously shown that deletion of cspA is associated with rapid conidial germination and reduced adhesion of dormant conidia. Here we show that cspA can be extracted with hydrofluoric acid (HF) from the cell wall, suggesting that it is a GPI-anchored CWP. The cspA-encoded CWP is unmasked during conidial germination and is surface expressed during hyphal growth. Deletion of cspA results in weakening of the conidial cell wall, whereas its overexpression increases conidial resistance to cell wall-degrading enzymes and inhibits conidial germination. Double mutant analysis indicates that cspA functionally interacts with the cell wall protein-encoding genes ECM33 and GEL2. Deletion of cspA together with ECM33 or GEL2 results in strongly reduced conidial adhesion, increased disorganization of the conidial cell wall, and exposure of the underlying layers of chitin and β-glucan. This is correlated with increasing susceptibility of the ΔcspA, ΔECM33, and ΔcspA ΔECM33 mutants to conidial phagocytosis and killing by human macrophages and hyphal damage induced by neutrophils. However, these strains did not exhibit altered virulence in mice with infected lungs. Collectively, these results suggest a role for cspA in maintaining the strength and integrity of the cell wall.
The critical event in heart formation is commitment of mesodermal cells to a cardiomyogenic fate, and cardiac fate determination is regulated by a series of cytokines. Bone morphogenetic proteins (BMPs) and fibroblast growth factors have been shown to be involved in this process, however additional factors needs to be identified for the fate determination, especially at the early stage of cardiomyogenic development.
Global gene expression analysis using a series of human cells with a cardiomyogenic potential suggested Gremlin (Grem1) is a candidate gene responsible for in vitro cardiomyogenic differentiation. Grem1, a known BMP antagonist, enhanced DMSO-induced cardiomyogenesis of P19CL6 embryonal carcinoma cells (CL6 cells) 10–35 fold in an area of beating differentiated cardiomyocytes. The Grem1 action was most effective at the early differentiation stage when CL6 cells were destined to cardiomyogenesis, and was mediated through inhibition of BMP2. Furthermore, BMP2 inhibited Wnt/β-catenin signaling that promoted CL6 cardiomyogenesis.
Grem1 enhances the determined path to cardiomyogenesis in a stage-specific manner, and inhibition of the BMP signaling pathway is involved in initial determination of Grem1-promoted cardiomyogenesis. Our results shed new light on renewal of the cardiovascular system using Grem1 in human.
Within the hierarchy of epithelial stem cells, normal progenitor cells may express regulated telomerase during renewal cycles of proliferation and differentiation. Dis-continuous telomerase activity may promote increased renewal capacity of progenitor cells, while deregulated/ continuous telomerase activity may promote immortalization when differentiation and/or senescent pathways are compromised. In the present work, we show that resveratrol activates, while progesterone inactivates, continuous telomerase activity within 24 h in subpopulations of human Li–Fraumeni syndrome-derived breast epithelial cells. Resveratrol results in immortalization of mixed progenitor cells with mutant p53, but not human epithelial cells with wild type p53. Our results demonstrate the potential for renewing progenitor cells with mutant p53 to immortalize after continuous telomerase expression when exposed to certain environmental compounds. Understanding the effects of telomerase modulators on endogenous telomerase activity in progenitor cells is relevant to the role of immortalization in the initiation and progression of cancer subtypes.
telomerase; p53; progesterone; breast epithelial; aging
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.
Many factors influence breast cancer progression, including the ability of progenitor cells to sustain or increase net tumour cell numbers. Our aim was to define whether alterations in putative progenitor populations could predict clinicopathological factors of prognostic importance for cancer progression.
Primary cultures were established from human breast tumour and adjacent non-tumour tissue. Putative progenitor cell populations were isolated based on co-expression or concomitant absence of the epithelial and myoepithelial markers EPCAM and CALLA respectively.
Significant reductions in cellular senescence were observed in tumour versus non-tumour cultures, accompanied by a stepwise increase in proliferation:senescence ratios. A novel correlation between tumour aggressiveness and an imbalance of putative progenitor subpopulations was also observed. Specifically, an increased double-negative (DN) to double-positive (DP) ratio distinguished aggressive tumours of high grade, estrogen receptor-negativity or HER2-positivity. The DN:DP ratio was also higher in malignant MDA-MB-231 cells relative to non-tumourogenic MCF-10A cells. Ultrastructural analysis of the DN subpopulation in an invasive tumour culture revealed enrichment in lipofuscin bodies, markers of ageing or senescent cells.
Our results suggest that an imbalance in tumour progenitor subpopulations imbalances the functional relationship between proliferation and senescence, creating a microenvironment favouring tumour progression.
Multiple progenitors derived from the heart and bone marrow have been utilized for cardiac repair. Despite this, not much is known about the molecular identity and relationship among these progenitors. To develop a robust stem cell therapy for the heart, it is critical to understand the molecular identity of the multiple ‘cardiogenic progenitor cells’ (CPCs). This study is the first report of high throughput transcriptional profiling of CPCs carried out on an identical platform.
Method and Results
Microarray based transcriptional profiling was carried out for three cardiac (ckit+, Sca1+, side population) and two bone marrow (ckit+ , mesenchymal stem cell) progenitors, obtained from age- and sex-matched wild type C57BL/6 mice. Analysis indicated that cardiac-derived ckit+ population was very distinct from Sca1+ and SP cells in the downregulation of genes encoding for cell-cell and matrix adhesion proteins, and in the upregulation of developmental genes. Significant enrichment of transcripts involved in DNA replication and repair was observed in bone marrow (BM)-derived progenitors. The BM ckit+ cells appeared to have the least correlation with the other progenitors, with enrichment of immature neutrophil specific molecules.
Our study indicates that cardiac ckit+ cells represent the most primitive population in the rodent heart. Primitive cells of cardiac versus BM origin differ significantly with respect to stemness and cardiac lineage-specific genes, and molecules involved in DNA replication and repair. The detailed molecular profile of progenitors reported here will serve as a useful reference to determine the molecular identity of progenitors used in future preclinical and clinical studies
cardiac progenitor cells; bone marrow cells; transcriptomics; cardiovascular diseases
Morphogenesis of the heart requires tight control of cardiac progenitor cell specification, expansion, and differentiation. Retinoic acid (RA) signaling restricts expansion of the second heart field (SHF), serving as an important morphogen in heart development. Here, we identify the LIM domain protein Ajuba as a crucial regulator of the SHF progenitor cell specification and expansion. Ajuba-deficient zebra-fish embryos show an increased pool of Isl1+ cardiac progenitors and, subsequently, dramatically increased numbers of cardiomyocytes at the arterial and venous poles. Furthermore, we show that Ajuba binds Isl1, represses its transcriptional activity, and is also required for autorepression of Isl1 expression in an RA-dependent manner. Lack of Ajuba abrogates the RA-dependent restriction of Isl1+ cardiac cells. We conclude that Ajuba plays a central role in regulating the SHF during heart development by linking RA signaling to the function of Isl1, a key transcription factor in cardiac progenitor cells.
When the gene for CspA, the major cold shock protein of Escherichia coli, was disrupted by a novel positive/negative selection method, the deltacspA cells did not show any discernible growth defect at either 37 or 15 degrees C. By two-dimensional gel electrophoresis, total protein synthesis was analyzed after temperature downshift in the deltacspA strain. The production of the CspA homologs CspB and CspG increased, and the duration of their expression was prolonged, suggesting that both CspB and CspG compensate for the function of CspA in the absence of CspA during cold shock adaptation. Interestingly, the production of the 159-base 5'-untranslated region (5'-UTR) of cspA from the chromosomal cspA::cat gene, detected by primer extension, failed to be repressed after cold shock. When an independent system to produce CspA was added to the deltacspA strain, the 5'-UTR production for the cspA::cat gene was significantly reduced compared to that of the deltacspA strain. By examining the expression of translationally fused cspA and cspB genes to lacZ in the deltacspA strain, it was found that cspA is more strongly regulated by CspA than cspB is. We showed that the increased expression of the 5'-UTR of the cspA mRNA in the deltacspA strain occurred mainly at the level of transcription and, to a certain extent, at the level of mRNA stabilization. The mRNA stabilization in the deltacspA strain was observed for other mRNAs, supporting the notion that CspA functions as an mRNA chaperone to destabilize secondary structures in mRNAs.