Chondro-osteogenic Differentiation Research Articles

Primary cilia mediate skeletogenic BMP and Hedgehog signaling in heterotopic ossification.

Heterotopic ossification (HO), defined as the formation of extraskeletal bone in muscle and soft tissues, is a diverse pathological process caused by either genetic mutations or inciting trauma. Fibrodysplasia ossificans progressiva (FOP) is a genetic form of HO caused by mutations in the bone morphogenetic protein (BMP) type I receptor gene activin A receptor type 1 (ACVR1). These mutations make ACVR1 hypersensitive to BMP and responsive to activin A. Hedgehog (Hh) signaling also contributes to HO development. However, the exact pathophysiology of how skeletogenic cells contribute to endochondral ossification in FOP remains unknown. Here, we showed that the wild-type or FOP-mutant ACVR1 localized in the cilia of stem cells from human exfoliated deciduous teeth with key FOP signaling components, including activin A receptor type 2A/2B, SMAD family member 1/5, and FK506-binding protein 12kD. Cilia suppression by deletion of intraflagellar transport 88 or ADP ribosylation factor like GTPase 3 effectively inhibited pathological BMP and Hh signaling, subdued aberrant chondro-osteogenic differentiation in primary mouse or human FOP cells, and diminished in vivo extraskeletal ossification in Acvr1Q207D, Sox2-Cre; Acvr1R206H/+ FOP mice and in burn tenotomy-treated wild-type mice. Our results provide a rationale for early and localized suppression of cilia in affected tissues after injury as a therapeutic strategy against either genetic or acquired HO.

Monotropein Protects Mesenchymal Stem Cells from Lipopolysaccharide-Induced Impairments and Promotes Fracture Healing in an Ovariectomized Mouse Model.

Monotropein is one of the active ingredients in Morinda Officinalis, which has been used for the treatment in multiple bone and joint diseases. This study aimed to observe the in vitro effects of Monotropein on osteogenic differentiation of lipopolysaccharide treated bone marrow mesenchymal stem cells (bMSCs), and the in vivo effects of local application of Monotropein on bone fracture healing in ovariectomized mice. Lipopolysaccharide was used to set up the inflammatory model in bMSCs, which were treated by Monotropein. Molecular docking analysis was performed to evaluate the potential interaction between Monotropein and p65. Transverse fractures of middle tibias were established in ovariectomized mice, and Monotropein was locally applied to the fracture site using injectable hydrogel. Monotropein enhanced the ability of primary bMSCs in chondro-osteogenic differentiation. Furthermore, Monotropein rescued lipopolysaccharide-induced osteogenic differentiation impairment and inhibited lipopolysaccharide-induced p65 phosphorylation in primary bMSCs. Docking analysis showed that the binding activity of Monotropein and p65/14-3-3 complex is stronger than the selective inhibitor of NF-κB (p65), DP-005. Local application of Monotropein partially rescued the decreased bone mass and biomechanical properties of callus or healed tibias in ovariectomized mice. The expressions of Runx2, Osterix and Collagen I in the 2-week callus were partially restored in Monotropein-treated ovariectomized mice. Taking together, local application of Monotropein promoted fracture healing in ovariectomized mice. Inhibition of p65 phosphorylation and enhancement in osteogenesis of mesenchymal stem cells could be partial of the effective mechanisms.

Mast4 determines the cell fate of MSCs for bone and cartilage development

Mesenchymal stromal cells (MSCs) differentiation into different lineages is precisely controlled by signaling pathways. Given that protein kinases play a crucial role in signal transduction, here we show that Microtubule Associated Serine/Threonine Kinase Family Member 4 (Mast4) serves as an important mediator of TGF-β and Wnt signal transduction in regulating chondro-osteogenic differentiation of MSCs. Suppression of Mast4 by TGF-β1 led to increased Sox9 stability by blocking Mast4-induced Sox9 serine 494 phosphorylation and subsequent proteasomal degradation, ultimately enhancing chondrogenesis of MSCs. On the other hand, Mast4 protein, which stability was enhanced by Wnt-mediated inhibition of GSK-3β and subsequent Smurf1 recruitment, promoted β-catenin nuclear localization and Runx2 activity, increasing osteogenesis of MSCs. Consistently, Mast4−/− mice demonstrated excessive cartilage synthesis, while exhibiting osteoporotic phenotype. Interestingly, Mast4 depletion in MSCs facilitated cartilage formation and regeneration in vivo. Altogether, our findings uncover essential roles of Mast4 in determining the fate of MSC development into cartilage or bone.

Degenerative changes of the aortic valve during left ventricular assist device support.

AimsDonor heart shortage leads to increasing use of left ventricular assist device (LVAD) as bridge‐to‐transplant or destination therapy. Prolonged LVAD support is associated with aortic valve insufficiency, representing a relevant clinical problem in LVAD patients. Nevertheless, the impact of LVAD support on inflammation, remodelling, and chondro‐osteogenic differentiation of the aortic valve is still not clearly understood. The aim of the study is to evaluate the impact of LVAD support on structural and molecular alterations of the aortic valve.Methods and resultsDuring heart transplantation, aortic valves of 63 heart failure patients without (n = 22) and with LVAD support (n = 41) were collected and used for analysis. Data on clinical course as well as echocardiographic data were analysed. Calcification and markers of remodelling, chondro‐osteogenic differentiation, and inflammation were evaluated by computed tomography, by mRNA analysis and by histology and immunohistochemistry. Expression of inflammation markers of the LVAD group was analysed with regard to levels of C‐reactive protein and driveline infections. Calcium accumulation and mRNA expression of determined markers were correlated with duration of LVAD support. Data were also analysed relating to aortic valve opening and aortic valve insufficiency. There was no difference in the frequency of cardiovascular risk factors or comorbidities between the patient groups. Expression of matrix metalloproteinase‐9 (P = 0.007), alpha‐smooth muscle actin (P = 0.045), and osteopontin (P = 0.003) were up‐regulated in aortic valves of LVAD patients. Histological appearance of the aortic valve was similar in patients with or without LVAD, and computed tomography‐based analysis not yet revealed significant difference in tissue calcification. Expression of interferon gamma (P = 0.004), interleukin‐1 beta (P < 0.0001), and tumour necrosis factor alpha (P = 0.04) was up‐regulated in aortic valves of LVAD patients without concomitant inflammatory cell infiltration and independent from unspecific inflammation. Expression of matrix metalloproteinase‐2 (P = 0.038) and transforming growth factor beta (P = 0.0504) correlated negatively with duration of LVAD support. Presence of aortic valve insufficiency led to a significantly higher expression of interferon gamma (P = 0.007) in LVAD patients. There was no alteration in the determined markers in relation to aortic valve opening in LVAD patients.ConclusionsLeft ventricular assist device support leads to signs of early aortic valve degeneration independent of support duration. Thus, the aortic valve of patients with LVAD support should be closely monitored, particularly in patients receiving destination therapy as well as in the prospect of using aortic valves of LVAD patients as homografts in case of bridge‐to‐transplant therapy.

ETS-Related Gene Expression in Healthy Femoral Arteries With Focal Calcifications.

Bone development-related genes are enriched in healthy femoral arteries, which are more prone to calcification, as documented by the predominance of fibrocalcific plaques at the femoral location. We undertook a prospective histological study on the presence of calcifications in normal femoral arteries collected from donors. Since endothelial-to-mesenchymal transition (EndMT) participates in vascular remodeling, immunohistochemical (IHC) and molecular markers of EndMT and chondro-osteogenic differentiation were assessed. Transmission electron microscopy (TEM) was used to describe calcification at its inception. Two hundred and fourteen femoral arteries were enrolled. The mean age of the donors was 39.9 ± 12.9 years; male gender prevailed (M: 128). Histology showed a normal architecture; calcifications were found in 52 (24.3%) cases, without correlations with cardiovascular risk factors. Calcifications were seen on or just beneath the inner elastic lamina (IEL). At IHC, SLUG was increasingly expressed in the wall of focally calcified femoral arteries (FCFA). ETS-related gene (ERG), SLUG, CD44, and SOX-9 were positive in calcifications. RT-PCR showed increased levels of BPM-2, RUNX-2, alkaline phosphatase, and osteocalcin osteogenic transcripts and increased expression of the chondrogenic marker, SOX-9, in FCFA. TEM documented osteoblast-like cells adjacent to the IEL, releasing calcifying vesicles from the cell membrane. The vesicles were embedded in a proteoglycan-rich matrix and were entrapped in IEL fenestrations. In this study, ERG- and CD44-positive cell populations were found in the context of increased SLUG expression, thus supporting the participation of EndMT in FCFA; the increased transcript expression of osteochondrogenic markers, particularly SOX-9, reinforced the view that EndMT, osteochondrogenesis, and neoangiogenesis interact in the process of arterial calcification. Given its role as a transcription factor in the regulation of endothelial homeostasis, arterial ERG expression can be a clue of endothelial dysregulation and changes in IEL organization which can ultimately hinder calcifying vesicle diffusion through the IEL fenestrae. These results may have a broader implication for understanding arterial calcification within a disease context.

Tissue Engineering Strategies to Increase Osteochondral Regeneration of Stem Cells; a Close Look at Different Modalities

The homeostasis of osteochondral tissue is tightly controlled by articular cartilage chondrocytes and underlying subchondral bone osteoblasts via different internal and external clues. As a correlate, the osteochondral region is frequently exposed to physical forces and mechanical pressure. On this basis, distinct sets of substrates and physicochemical properties of the surrounding matrix affect the regeneration capacity of chondrocytes and osteoblasts. Stem cells are touted as an alternative cell source for the alleviation of osteochondral diseases. These cells appropriately respond to the physicochemical properties of different biomaterials. This review aimed to address some of the essential factors which participate in the chondrogenic and osteogenic capacity of stem cells. Elements consisted of biomechanical forces, electrical fields, and biochemical and physical properties of the extracellular matrix are the major determinant of stem cell differentiation capacity. It is suggested that an additional certain mechanism related to signal-transduction pathways could also mediate the chondro-osteogenic differentiation of stem cells. The discovery of these clues can enable us to modulate the regeneration capacity of stem cells in osteochondral injuries and lead to the improvement of more operative approaches using tissue engineering modalities.

Transcriptomic profiling of experimental arterial injury reveals new mechanisms and temporal dynamics in vascular healing response

ObjectiveEndovascular interventions cause arterial injury and induce a healing response to restore vessel wall homeostasis. Complications of defective or excessive healing are common and result in increased morbidity and repeated interventions. Experimental models of intimal hyperplasia are vital for understanding the vascular healing mechanisms and resolving the clinical problems of restenosis, vein graft stenosis, and dialysis access failure. Our aim was to systematically investigate the transcriptional, histologic, and systemic reaction to vascular injury during a prolonged time. MethodsBalloon injury of the left common carotid artery was performed in male rats. Animals (n = 69) were euthanized before or after injury, either directly or after 2 hours, 20 hours, 2 days, 5 days, 2 weeks, 6 weeks, and 12 weeks. Both injured and contralateral arteries were subjected to microarray profiling, followed by bioinformatic exploration, histologic characterization of the biopsy specimens, and plasma lipid analyses. ResultsImmune activation and coagulation were key mechanisms in the early response, followed by cytokine release, tissue remodeling, and smooth muscle cell modulation several days after injury, with reacquisition of contractile features in later phases. Novel pathways related to clonal expansion, inflammatory transformation, and chondro-osteogenic differentiation were identified and immunolocalized to neointimal smooth muscle cells. Analysis of uninjured arteries revealed a systemic component of the reaction after local injury, underlined by altered endothelial signaling, changes in overall tissue bioenergy metabolism, and plasma high-density lipoprotein levels. ConclusionsWe demonstrate that vascular injury induces dynamic transcriptional landscape and metabolic changes identifiable as early, intermediate, and late response phases, reaching homeostasis after several weeks. This study provides a temporal “roadmap” of vascular healing as a publicly available resource for the research community.

Glucose regulates tissue-specific chondro-osteogenic differentiation of human cartilage endplate stem cells via O-GlcNAcylation of Sox9 and Runx2

BackgroundThe degenerative disc disease (DDD) is a major cause of low back pain. The physiological low-glucose microenvironment of the cartilage endplate (CEP) is disrupted in DDD. Glucose influences protein O-GlcNAcylation via the hexosamine biosynthetic pathway (HBP), which is the key to stem cell fate. Thiamet-G is an inhibitor of O-GlcNAcase for accumulating O-GlcNAcylated proteins while 6-diazo-5-oxo-l-norleucine (DON) inhibits HBP. Mechanisms of DDD are incompletely understood but include CEP degeneration and calcification. We aimed to identify the molecular mechanisms of glucose in CEP calcification in DDD.MethodsWe assessed normal and degenerated CEP tissues from patients, and the effects of chondrogenesis and osteogenesis of the CEP were determined by western blot and immunohistochemical staining. Cartilage endplate stem cells (CESCs) were induced with low-, normal-, and high-glucose medium for 21 days, and chondrogenic and osteogenic differentiations were measured by Q-PCR, western blot, and immunohistochemical staining. CESCs were induced with low-glucose and high-glucose medium with or without Thiamet-G or DON for 21 days, and chondrogenic and osteogenic differentiations were measured by Q-PCR, western blot, and immunohistochemical staining. Sox9 and Runx2 O-GlcNAcylation were measured by immunofluorescence. The effects of O-GlcNAcylation on the downstream genes of Sox9 and Runx2 were determined by Q-PCR and western blot.ResultsDegenerated CEPs from DDD patients lost chondrogenesis, acquired osteogenesis, and had higher protein O-GlcNAcylation level compared to normal CEPs from LVF patients. CESC chondrogenic differentiation gradually decreased while osteogenic differentiation gradually increased from low- to high-glucose differentiation medium. Furthermore, Thiamet-G promoted CESC osteogenic differentiation and inhibited chondrogenic differentiation in low-glucose differentiation medium; however, DON acted opposite role in high-glucose differentiation medium. Interestingly, we found that Sox9 and Runx2 were O-GlcNAcylated in differentiated CESCs. Finally, O-GlcNAcylation of Sox9 and Runx2 decreased chondrogenesis and increased osteogenesis in CESCs.ConclusionsOur findings demonstrate the effect of glucose concentration on regulating the chondrogenic and osteogenic differentiation potential of CESCs and provide insight into the mechanism of how glucose concentration regulates Sox9 and Runx2 O-GlcNAcylation to affect the differentiation of CESCs, which may represent a target for CEP degeneration therapy.

Alteration of the Total Cellular Glycome during Late Differentiation of Chondrocytes

In normal articular cartilage, chondrocytes do not readily proliferate or terminally differentiate, and exhibit a low level of metabolism. Hypertrophy-like changes of chondrocytes have been proposed to play a role in the pathogenesis of osteoarthritis by inducing protease-mediated cartilage degradation and calcification; however, the molecular mechanisms underlying these changes are unclear. Glycans are located on the outermost cell surface. Dynamic cellular differentiation can be monitored and quantitatively characterized by profiling the glycan structures of total cellular glycoproteins. This study aimed to clarify the alterations in glycans upon late differentiation of chondrocytes, during which hypertrophy-like changes occur. Primary mouse chondrocytes were differentiated using an insulin-induced chondro-osteogenic differentiation model. Comprehensive glycomics, including N-glycans, O-glycans, free oligosaccharides, glycosaminoglycan, and glycosphingolipid, were analyzed for the chondrocytes after 0-, 10- and 20-days cultivation. The comparison and clustering of the alteration of glycans upon hypertrophy-like changes of primary chondrocytes were performed. Comprehensive glycomic analyses provided complementary alterations in the levels of various glycans derived from glycoconjugates during hypertrophic differentiation. In addition, expression of genes related to glycan biosynthesis and metabolic processes was significantly correlated with glycan alterations. Our results indicate that total cellular glycan alterations are closely associated with chondrocyte hypertrophy and help to describe the glycophenotype by chondrocytes and their hypertrophic differentiation. our results will assist the identification of diagnostic and differentiation biomarkers in the future.

Overexpression of mechanical sensitive miR-337-3p alleviates ectopic ossification in rat tendinopathy model via targeting IRS1 and Nox4 of tendon-derived stem cells.

Tendinopathy, which is characterized by the ectopic ossification of tendon, is a common disease occurring in certain population, such as athletes that suffer from repetitive tendon strains. However, the molecular mechanism underlying the pathogenesis of tendinopathy caused by the overuse of tendon is still lacking. Here, we found that the mechanosensitive miRNA, miR-337-3p, had lower expression under uniaxial cyclical mechanical loading in tendon-derived stem cells (TDSCs) and negatively controlled chondro-osteogenic differentiation of TDSCs. Importantly, downregulation of miR-337-3p expression was also observed in both rat and human calcified tendons, and overexpressing miR-337-3p in patellar tendons of rat tendinopathy model displayed a robust therapeutic efficiency. Mechanistically, we found that the proinflammatory cytokine interleukin-1β was the upstream factor of miR-337-3p that bridges the mechanical loading with its downregulation. Furthermore, the target genes of miR-337-3p, NADPH oxidase 4, and insulin receptor substrate 1, activated chondro-osteogenic differentiation of TDSCs through JNK and ERK signaling, respectively. Thus, these findings not only provide novel insight into the molecular mechanisms underlying ectopic ossification in tendinopathy but also highlight the significance of miR-337-3p as a putative therapeutic target for clinic treatment of tendinopathy.

Overexpression of Mechanical Sensitive miR-337-3p Alleviates Ectopic Ossification in Rat Tendinopathy Model Via Targeting IRS1 and Nox4 of Tendon Derived Stem Cells

Tendinopathy, which is characterized by the ectopic ossification of tendon, is a common disease occurred in population, such as athletes that suffer from repetitive tendon strains. However, the molecular mechanism underlying the pathogenesis of tendinopathy caused by overuse of tendon is still lacking. Here, we found that the mechanosensitive miRNA, miR-337-3p, reduces its expression under uniaxial cyclical mechanical loading in tendon-derived stem cells (TDSCs) and negatively controlled chondro-osteogenic differentiation of TDSCs. Importantly, down-regulation of miR-337-3p expression was also observed in both rat and human calcified tendons, and overexpressing miR-337-3p in patellar tendons of rat tendinopathy model displayed a robust therapeutic efficiency. Mechanistically, we found that the proinflammatory cytokine Interleukin-1β (IL-1β) was the upstream factor of miR-337-3p that bridges the mechanical loading with its down-regulation. Furthermore, the target genes of miR-337-3p, NADPH oxidase 4 (Nox4) and insulin receptor substrate 1 (IRS1) activated chondro-osteogenic differentiation of TDSCs through JNK and ERK signaling respectively. Thus, these findings not only provide novel insight into the molecular mechanisms underlying ectopic ossification in tendinopathy but also highlight the significance of miR-337-3p as a putative therapeutic target for clinic treatment of tendinopathy. Funding: This work was supported by grants from National Natural Science Foundation of China (No.81572123, 81401844), The Ministry of Science and Technology of China (No. 2015DFG32200), Science and Technology Commission of Shanghai Municipality (No.14431900900, 15411951100, 16430723500), Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (No.20161314). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: All experiments with animals were carried according to Experimental Safety of Chinese Academy of Sciences and the Committees of Animal Ethics. All animal treatments were guided by the NIH guidelines.

A genome-wide analysis of the gene expression profiles and alternative splicing events during the hypoxia-regulated osteogenic differentiation of human cartilage endplate-derived stem cells.

It has been hypothesized that intervertebral disc degeneration is initiated by degeneration of the cartilage endplate (CEP), which is characterized by cartilage ossification. CEP-derived stem cells (CESCs), with the potential for chondro-osteogenic differentiation, may be responsible for the balance between chondrification and ossification in the CEP. The CEP remains in an avascular and hypoxic microenvironment; the present study observed that hypoxia was able to markedly inhibit the osteogenic differentiation of CESCs. This tissue-specific CESC differentiation in response to a hypoxic microenvironment was physiologically important for the prevention of ossification in the CEP. In order to study the hypoxia-regulated mechanisms underlying osteogenic differentiation of CESCs, a Human Transcriptome Array 2.0 was used to detect differentially expressed genes (DEGs) and alternatively spliced genes (ASGs) during the osteogenic differentiation of CESCs under hypoxia, compared with those induced under normoxia. High-throughput analysis of DEGs and ASGs demonstrated that genes in the complement pathway were enriched, which may be a potential mechanism underlying hypoxia inhibition of CESCs osteogenesis. The results of the present study may provide a basis for future mechanistic studies regarding gene expression levels and alternative splicing events during the hypoxia-regulated inhibition of osteogenesis, which may be helpful in identifying targets for CEP degeneration therapy.

MIF Plays a Key Role in Regulating Tissue-Specific Chondro-Osteogenic Differentiation Fate of Human Cartilage Endplate Stem Cells under Hypoxia.

SummaryDegenerative cartilage endplate (CEP) shows decreased chondrification and increased ossification. Cartilage endplate stem cells (CESCs), with the capacity for chondro-osteogenic differentiation, are responsible for CEP restoration. CEP is avascular and hypoxic, while the physiological hypoxia is disrupted in the degenerated CEP. Hypoxia promoted chondrogenesis but inhibited osteogenesis in CESCs. This tissue-specific differentiation fate of CESCs in response to hypoxia was physiologically significant with regard to CEP maintaining chondrification and refusing ossification. MIF, a downstream target of HIF1A, is involved in cartilage and bone metabolisms, although little is known about its regulatory role in differentiation. In CESCs, MIF was identified as a key point through which HIF1A regulated the chondro-osteogenic differentiation. Unexpectedly, unlike the traditionally recognized mode, increased nuclear-expressed MIF under hypoxia was identified to act as a transcriptional regulator by interacting with the promoter of SOX9 and RUNX2. This mode of HIF1A/MIF function may represent a target for CEP degeneration therapy.

Sequential differentiation of human bone marrow stromal cells for bone regeneration

In this study we hypothesized that as a simulation of endochondral bone formation, bone marrow stromal cell (BMSC) provide a sequential chondro-osteogenic differentiation potential. A chondrogenic priming of BMSC leads to a spontaneous three-dimensional cell formation. BMSC were chondrogenically differentiated prior to an osteogenic stimulation. Duration of cell culture was 28 days, whereas in group A BMSC were chondrogenically differentiated for 1 day, followed by an osteogenic differentiation for 27 days. In group B BMSC were chondrogenically differentiated for 14 days prior to an osteogenic differentiation of 14 days and group C BMSC were differentiated chondrogenically for 28 days serving as a chondrogenic control group. Chondrogenic priming induced a spontaneous three-dimensional cell formation. To survey the stability of the osteogenic phenotype in the absence of an osteogenic stimulus, investigations were performed in vivo in a specially adapted chorioallantoic membrane model of fertilized White Leghorn eggs. Histology and real time polymerase chain reaction revealed a higher amount of osteogenic extracellular matrix synthesis and significant higher expressions of osteogenic marker genes in group B after 14 days of chondrogenic and 14 days of osteogenic stimulation. Matrix calcification in vivo in the absence of an osteogenic stimulus could be demonstrated. The results of the present study support the theory of a sequential differentiation potential of BMSC. A chondrogenic priming of BMSC stimulated into the osteogenic lineage result in a stable osteogenic phenotype in a scaffold-free, three-dimensional tissue engineering application.

Arterial calcification: Finger-pointing at resident and circulating stem cells.

The term ''Stammzelle'' (stem cells) originally appeared in 1868 in the works of Ernst Haeckel who used it to describe the ancestor unicellular organism from which he presumed all multicellular organisms evolved. Since then stem cells have been studied in a wide spectrum of normal and pathological conditions; it is remarkable to note that ectopic arterial calcification was considered a passive deposit of calcium since its original discovering in 1877; in the last decades, resident and circulating stem cells were imaged to drive arterial calcification through chondro-osteogenic differentiation thus opening the idea that an active mechanism could be at the basis of the process that clinically shows a Janus effect: calcifications either lead to the stabilization or rupture of the atherosclerotic plaques. A review of the literature underlines that 130 years after stem cell discovery, antigenic markers of stem cells are still debated and the identification of the osteoprogenitor phenotype is even more elusive due to tissue degradation occurring at processing and manipulation. It is necessary to find a consensus to perform comparable studies that implies phenotypic recognition of stem cells antigens. A hypothesis is based on the singular morphology and amitotic mechanism of division of osteoclasts: it constitutes the opening to a new approach on osteoprogenitors markers and recognition. Our aim was to highlight all the present evidences of the active calcification process, summarize the different cellular types involved, and discuss a novel approach to discover osteoprogenitor phenotypes in arterial wall.

Calcification after myocardial infarction is independent of amniotic fluid stem cell injection

Ischemic heart disease remains one of the most common causes of mortality in developed countries. Recently, stem cell therapy is being considered for treating ischemic heart diseases. On the other hand, there has been evidence of chondro-osteogenic mass formation after stem cell injection in the heart. In a recent publication, Chiavegato et al. (J Mol Cell Cardiol. 42 (2007) 746-759) has suggested that amniotic fluid-derived stem (AFS) cells cause chondro-osteogenic masses in the infarcted heart. The goal of the current study was to further examine the formation of such masses, specifically, the role of AFS cells in this process. Our results confirm the presence of similar bone-like masses in the left ventricular wall of infarcted rats; however, this phenomenon occurred independent of AFS cell injection into the myocardium. Moreover, AFS cell injection did not increase the presence of chondro-osteogenic masses. Echocardiographic analysis of large infarctions in rats frequently revealed the presence of echogenic structures in the left ventricular wall. We further demonstrated a significant relationship between the infarction size and chondro-osteogenic formation and subsequent decrease in cardiac function. Collectively, our study indicates that chondro-osteogenic differentiation can take place in infarcted rat heart independent of cell injection. These results have significant implications for future design and testing of stem cell therapy for treatment of cardiac muscle diseases.

Inhibition of bone morphogenetic protein protects against atherosclerosis and vascular calcification

The bone morphogenetic proteins (BMP), a family of morphogens, have been implicated in atherogenesis as mediators of inflammation and calcification. We investigated the role of altered expression of Matrix Gla Protein (MGP), an inhibitor of BMP, on the development of atherosclerotic lesions and vascular calcification using MGP transgenic or MGP deficient mice bred to apolipoprotein E (Apoe−/−) mice, a model of atherosclerosis. MPG overexpression reduced vascular BMP signaling, which resulted in reduced lesion size, and intimal and medial calcification. It was associated with reduced expression of the activin‐like kinase receptor (ALK)1 and the vascular endothelial growth factor (VEGF), part of a BMP‐activated pathway that also regulates angiogenesis. MGP‐deficiency, on the other hand, strongly enhanced vascular BMP signaling and expression of ALK1 and VEGF. Despite the high propensity of the Apoe−/− mice to develop lesions, lesion formation was minimal in the setting of MGP deficiency, which causes chondroosteogenic differentiation and calcification, suggesting that the abnormal cell differentiation did not allow for lesion formation. Together, our results indicate that vascular BMP signaling is a key regulator in the development of vascular disease, requiring careful control to maintain normal vascular homeostasis.Research support: NIH/NHLBI, AHA.

Human amniotic fluid-derived stem cells are rejected after transplantation in the myocardium of normal, ischemic, immuno-suppressed or immuno-deficient rat

Human amniotic fluid-derived stem (AFS) cells, similarly to embryonic stem cells, could possess privileged immunological characteristics suitable for a successful transplantation even in a discordant xenograft system. We investigated whether AFS cells could be fruitfully used in a rat model of myocardial infarction. c-kit immunomagnetic-sorted AFS cells were characterized by flow cytometric analysis and cytospins as well as reverse-transcription polymerase chain reaction, Western blotting and immunocytochemistry for cardiovascular differentiation markers. In vitro, AFS cell phenotypic conversion was assayed by cardiovascular-specific induction media or co-cultured with rat neonatal cardiomyocytes. AFS cells showed mRNAs and/or protein for endothelial (angiopoietin, CD146) and smooth muscle (smoothelin) cells, and cardiomyocyte (Nkx2.5, MLC-2v, GATA-4, β-MyHC) markers. Acquisition of a cardiomyocyte-like phenotype in rare AFS cells could be seen only in co-cultures with rat neonatal cells. In vivo, AFS cells xenotransplantated in a rat model of myocardial infarction, with or without cyclosporine treatment, or in intact heart from immuno-competent or immuno-deficient animals were acutely rejected due to the different recruitment of recipient CD4 +, CD8 + T and B lymphocytes, NK cells and macrophages. This reaction is most likely to be linked to expression of B7 co-stimulatory molecules CD80 and CD86 as well as macrophage marker CD68 on AFS cells. Xenotransplanted AFS cells gave also rise in some animals to cell masses in the subendocardium and myocardium suggestive of a process of chondro-osteogenic differentiation. Despite AFS cells in vitro can differentiate to some extent to cells of cardiovascular lineages, their in vivo use in xenotransplantation for cell therapy of myocardial infarction is hampered by their peculiar immunogenic properties and phenotypic instability.

Ectopic Ossification in the Scar Tissue of Rats with Myocardial Infarction.

We describe the occurrence of bone-like formations in the left ventricular wall of infarcted rats treated or not with bone marrow cells injected systemically or locally into the myocardium. The incidence of ectopic calcification in hearts has been reported in rare cases in children with infarcts without previous coronary artery disease. Recently, ventricular calcification has been correlated with unselected bone marrow cell transplantation into infarcted rat hearts. Echocardiographic analysis of large infarction in rats frequently reveals the presence of echogenic structures in the left ventricular wall, sometimes projecting to the lumen of the chamber. The histological examination of these echogenic structures exhibited bone, cartilage, and marrowlike formations extending from the collagen-rich matrix of the ventricle wall. Microanalytical techniques verified the presence of hydroxyapatite in the mineral phase. Ossification was found in 25 out of 30 hearts evaluated 90 days postinfarct, being observed in 14 out of 17 animals submitted to cell therapy and in 11 out of 13 infarcted rats not submitted to cell therapy. Our study indicates that chondro-osteogenic differentiation can take place in the pathological rat heart independent of animal treatment with marrow cells.

Formation of a chondro-osseous rudiment in micromass cultures of human bone-marrow stromal cells.

Bone-marrow stromal cells can differentiate into multiple mesenchymal lineages including cartilage and bone. When these cells are seeded in high-density 'pellet culture', they undergo chondrogenesis and form a tissue that is morphologically and biochemically defined as cartilage. Here, we show that dual chondro-osteogenic differentiation can be obtained in the same micromass culture of human bone-marrow stromal cells. Human bone-marrow stromal cells were pellet cultured for 4 weeks in chondro-inductive medium. Cartilage 'beads' resulting from the micromass culture were then subcultured for further 1-3 weeks in osteo-inductive medium. This resulted in the formation of a distinct mineralized bony collar around hyaline cartilage. During the chondrogenesis phase, type I collagen and bone sialoprotein were produced in the outer portion of the cartilage bead, which, upon subsequent exposure to beta-glycerophosphate, mineralized and accumulated extracellular bone sialoprotein and osteocalcin. Our modification of the pellet culture system results in the formation of a chondro-osseous 'organoid' structurally reminiscent of pre-invasion endochondral rudiments, in which a bony collar forms around hyaline cartilage. The transition from a cell culture to an organ culture dimension featured by our system provides a suitable model for the dissection of molecular determinants of endochondral bone formation, which unfolds in a precisely defined spatial and temporal frame