Ultrasound combined with glial cell line-derived neurotrophic factor-loaded microbubbles for the targeted treatment of drug addiction.

Drug addiction is a serious problem globally, recently exacerbated by the COVID-19 pandemic. Glial cell-derived neurotrophic factor (GDNF) is considered a potentially effective strategy for the treatment of addiction. Previous animal experiments have proven that GDNF has a good therapeutic effect on drug addiction, but its clinical application is limited due to its poor blood-brain barrier (BBB) permeability. Low-frequency focused ultrasound, combined with microbubbles, is a non-invasive and reversible technique for locally-targeted BBB opening. In the present study, magnetic resonance imaging-guided low-frequency focused ultrasound, combined with GDNF microbubbles, was used to target BBB opening in the ventral tegmental area (VTA) region. The effects of GDNF on morphine-induced conditioned place preference (CPP) and acute withdrawal symptoms in rats after a partially opened BBB were evaluated by behavioral observation. Western blot was used to detect changes in tyrosine hydroxylase (TH) expression levels in the VTA region after different treatments, and high performance liquid chromatography was used to detect the changes in monoamine neurotransmitter content. The results showed that ultrasound combined with GDNF microbubbles targeted and opened the BBB in the VTA region, and significantly increased GDNF content, destroyed morphine-induced CPP, and reduced the withdrawal symptoms of morphine addiction in rats. Furthermore, the up-regulation of TH expression and the increase of norepinephrine and dopamine content induced by morphine were significantly reversed, and the increase of 5-hydroxytryptamine content was partially reversed. Therefore, ultrasound combined with GDNF microbubbles to target and open the BBB can effectively increase the content of central GDNF, thus playing a therapeutic role in morphine addiction. Our study provides a new approach to locally open the BBB and target delivery of neurotrophic factors, such as GDNF, to treat brain diseases like addiction.

Therapeutic Effect of Ultrasound Combined With Porous Lipid Clioquinol/PLGA Microbubbles on Ferroptosis in HL-1 Cardiac Cell Induced by Isoproterenol Attack.

In recent years, studies have shown a close relationship between cardiomyocyte death and ferroptosis. Clioquinol (CQ) can inhibit ferroptosis. Porous lipid-poly (lactic-co-glycolic acid) (PLGA) microbubbles (MBs) were prepared by double emulsification (W1/O/W2) using 1,2-dioctadecanoyl-sn-glycero-3-phophocholine and PLGA as raw materials. Porous lipid-PLGA MBs were used as carriers to prepare CQ/PLGA MBs containing CQ. CQ/PLGA had the advantages of high drug loading, good biocompatibility, and sustained release. Our results showed that CQ/PLGA improved the effect of CQ and reduced its cytotoxicity. Under low-frequency ultrasound with certain parameters, CQ/PLGA showed steady-state cavitation, which increased the membrane permeability of mouse cardiomyocyte HL-1 to a certain extent and further prevented the process of ferroptosis in mouse cardiomyocyte HL-1.

Ultrasound-triggered drug delivery for glioma therapy through gambogic acid-loaded nanobubble-microbubble complexes.

Glioma is one of the most common primary brain tumors. Gambogic acid (GA) is widely used in tumor chemotherapy. However, GA has poor water solubility, low bioavailability, and difficult permeability across the blood-brain barrier (BBB), leading to poor efficacy against brain tumors. In our study, we developed negatively charged GA-loaded PLGA nanobubbles [GA/poly(lactic-co-glycolic acid) (PLGA)] and conjugated them onto the surface of cationic lipid microbubbles (CMBs) through electrostatic interactions. The resulting GA/PLGA-CMB complex was characterized for its particle size, distribution, drug encapsulation efficiency, and ultrasound imaging property, revealing a high drug encapsulation efficiency and excellent contrast imaging capability. Importantly, significantly enhanced GA delivery into the brain could be observed after the intravenous administration of GA/PLGA-CMBs combined with low-intensity focused ultrasound (FUS) due to the cavitation from CMBs, which mediated blood-brain barrier (BBB) opening. Taking advantage of the opened BBB, GA/PLGA nanobubbles could be delivered into the tumor. Then, the second FUS irradiation at higher energy was used to induce the cavitation of GA/PLGA nanobubbles, producing the second cavitation on tumor cells, significantly enhancing the ability of GA to enter tumor cells and inhibit tumor growth inhibition efficacy.

A Gambogic Acid-Loaded Delivery System Mediated by Ultrasound-Targeted Microbubble Destruction: A Promising Therapy Method for Malignant Cerebral Glioma.

Background: The blood-brain barrier (BBB) inhibits the delivery of macromolecular chemotherapeutic drugs to brain tumors, leading to low utilization rates and toxic side effects to surrounding tissues and organs. Ultrasonic targeted microbubble destruction (UTMD) technology can open the BBB, leading to a new type of drug delivery system with particular utility in glioma. Purpose: We have developed a new type of drug-loaded microbubble complex based on poly(lactic-co-glycolic acid) (PLGA) that targets gambogic acid (GA) to the area of brain tumors through UTMD. Methods: GA/PLGA nanoparticles were prepared by the double emulsification method, and cationic microbubbles (CMBs) were prepared by a thin film hydration method. The GA/PLGA-CMB microbubble complex was assembled through electrostatic attractions and was characterized chemically. The anti-glioblastoma effect of GA/PLGA-CMB combined with focused ultrasound (FUS) was evaluated by biochemical and imaging assays in cultured cells and model mice. Results: GA/PLGA-CMB combined with FUS demonstrated a significant inhibitory effect on glioblastoma cell lines U87 and U251 as compared with controls (P<0.05). Tumor access and imaging analyses demonstrated that administration of GA/PLGA-CMBs combined with FUS can open the BBB and target the treatment of glioblastoma in a mouse model, as compared with control groups (P<0.05). Conclusion: The combination of PLGA-CMB with FUS provides an effective and biocompatible drug delivery system, and its application to the delivery of GA in a mouse glioblastoma model was successful.

The sustained PGE2 release matrix improves neovascularization and skeletal muscle regeneration in a hindlimb ischemia model.

BACKGROUND: The promising therapeutic strategy for the treatment of peripheral artery disease (PAD) is to restore blood supply and promote regeneration of skeletal muscle regeneration. Increasing evidence revealed that prostaglandin E2 (PGE2), a lipid signaling molecule, has significant therapeutic potential for tissue repair and regeneration. Though PGE2 has been well reported in tissue regeneration, the application of PGE2 is hampered by its short half-life in vivo and the lack of a viable system for sustained release of PGE2. RESULTS: In this study, we designed and synthesized a new PGE2 release matrix by chemically bonding PGE2 to collagen. Our results revealed that the PGE2 matrix effectively extends the half-life of PGE2 in vitro and in vivo. Moreover, the PGE2 matrix markedly improved neovascularization by increasing angiogenesis, as confirmed by bioluminescence imaging (BLI). Furthermore, the PGE2 matrix exhibits superior therapeutic efficacy in the hindlimb ischemia model through the activation of MyoD1-mediated muscle stem cells, which is consistent with accelerated structural recovery of skeletal muscle, as evidenced by histological analysis. CONCLUSIONS: Our findings highlight the chemical bonding strategy of chemical bonding PGE2 to collagen for sustained release and may facilitate the development of PGE2-based therapies to significantly improve tissue regeneration.

Renal subcapsular delivery of PGE2 promotes kidney repair by activating endogenous Sox9+ stem cells.

Prostaglandin E2 (PGE2) has recently been recognized to play a role in immune regulation and tissue regeneration. However, the short half-life of PGE2 limits its clinical application. Improving the delivery of PGE2 specifically to the target organ with a prolonged release method is highly desirable. Taking advantage of the adequate space and proximity of the renal parenchyma, renal subcapsular delivery allows minimally invasive and effective delivery to the entire kidney. Here, we report that by covalently cross-linking it to a collagen matrix, PGE2 exhibits an adequate long-term presence in the kidney with extensive intraparenchymal penetration through renal subcapsular delivery and significantly improves kidney function. Sox9 cell lineage tracing with intravital microscopy revealed that PGE2 could activate the endogenous renal progenitor Sox9+ cells through the Yap signaling pathway. Our results highlight the prospects of utilizing renal subcapsular-based drug delivery and facilitate new applications of PGE2-releasing matrices for regenerative therapy.

Effect of Gambogic Acid-Loaded Porous-Lipid/PLGA Microbubbles in Combination With Ultrasound-Triggered Microbubble Destruction on Human Glioma.

Gambogic acid (GA) is a highly effective antitumor agent, and it is used for the treatment of a wide range of cancers. It is challenging to deliver drugs to the central nervous system due to the inability of GA to cross the blood-brain barrier (BBB). Studies have shown that ultrasound-targeted microbubble destruction can be used for transient and reversible BBB disruption, significantly facilitating intracerebral drug delivery. We first prepared GA-loaded porous-lipid microbubbles (GA porous-lipid/PLGA MBs), and an in vitro BBB model was established. The cell viability was detected by CCK-8 assay and flow cytometry. The results indicate that U251 human glioma cells were killed by focused ultrasound (FUS) combined with GA/PLGA microbubbles. FUS combined with GA/PLGA microbubbles was capable of locally and transiently enhancing the permeability of BBB under certain conditions. This conformational change allows the release of GA to extracellular space. This study provides novel targets for the treatment of glioma.

[Expression of ANO1 during cardiac fibroblasts differentiation and its electrophysiological characteristics as calcium-activated chloride channel protein].

OBJECTIVE: To investigate the expression and electrophysiological characteristics of calcium-activated chlorine channel anoctamin-1 (ANO1) protein during the differentiation of cardiac fibroblasts (CFs) into myofibroblasts (MFs), and to elucidate the role of ANO1 in myocardial fibrosis. METHODS: The primary CFs from neonatal rats were isolated and the cells differentiated into MFs by subculture. The Ca2+-activated Cl- current (ICl(Ca)) in CFs and MFs were measured by whole-cell patch clamp, and the expressions of ANO1, α-smooth muscle actin(α-SMA)and vimentin in CFs and MFs were detected by immunofluorescence assay and Western blot, respectively. RESULTS: The current density in the early adherent CFs was stronger than that in MFs. ANO1 was expressed preferentially within and around the nuclei, and a small amount of ANO1 was expressed on the cell membrane. Moreover, ANO1 expression was weak in the early adherent CFs and displayed stronger expression in the MFs with proliferation tendency. CONCLUSION: The expression of ANO1 is closely related to the differentiation of MFs and it may be involved in modulation myocardial fibrosis.

ANO1 regulates cardiac fibrosis via ATI-mediated MAPK pathway.

Cardiac fibrosis is associated with most of heart diseases, but its molecular mechanism remains unclear. Anoctamin-1 (ANO1), a calcium-activated chloride channels (CaCCs) protein, plays a critical role in various pathophysiological processes. In the current study, we identified ANO1 expression in myocardial infarction (MI) model of rat and verified the role of ANO1 in cardiac fibrosis using transcriptomics combined with RNAi assays. we found that ANO1 expression was increased during the first two weeks, and decreased in the third week after MI. Fluorescence double labeling showed that ANO1 was mainly expressed in cardiac fibroblasts (CFs) and displayed an increased expression in CFs with proliferation tendency. The proliferation and secretion of CFs were markedly inhibited by knockdown of ANO1. RNA-Seq showed that most of the downregulation genes were related to the proliferation of CFs and cardiac fibrosis. After ANO1 knockdown, the expressions of angiotensin II type 1 receptor (AT1R) and cell nuclear proliferation antigen were markedly reduced, and the phosphorylation levels of MEK and ERK1/2 was decreased significantly, indicating that ANO1 regulate cardiac fibrosis through ATIR-mediated MAPK signaling pathway. These findings would be useful for the development of therapeutic strategies targeting ANO1 to treat and prevent cardiac fibrosis.

IGF-1C domain-modified chitosan hydrogel accelerates cutaneous wound healing by promoting angiogenesis.

Background: Complete regeneration after skin injury remains a critical clinical challenge. Hydrogels, modified with growth factors or mimicking peptides, have been applied for functional tissue regeneration by increasing the bioactivity of engineered matrices. Methodology & results: We synthesized an injectable biological hydrogel, C domain of IGF-1 (IGF-1C)-modified chitosan (CS-IGF-1C) hydrogel. Mouse model of cutaneous wound healing was established to investigate whether this hydrogel could promote wound healing. Our results demonstrated that CS-IGF-1C hydrogel exhibited superior proangiogenic effects, resulting in accelerated wound closure and improved extracellular matrix remodeling. Bioluminescence imaging and histology analysis confirmed the proangiogenic role of CS-IGF-1C hydrogel. Conclusion: CS-IGF-1C hydrogel could accelerate cutaneous wound healing by stimulating angiogenesis.

Inhibition of profibrotic signalling enhances the 5-azacytidine-induced reprogramming of fibroblasts into cardiomyocytes.

Recent studies have shown that cardiac fibroblasts (CFs) can be transformed into induced cardiomyocytes (iCMs). This phenomenon represents a potential method for rescuing damaged myocardia after myocardial infarction. The mechanism underlying cardiac reprogramming regulation must be clarified to improve the induction efficiency of iCMs. In this study, we treated CFs with 5-aza for 24 h and added TGF-ß inhibitor A83-01 for 2 weeks in vitro to investigate the effect of inhibiting fibrosis on myocardial differentiation. Inhibition of TGF-ß1 activity with A83-01 significantly decreased the expressions of collagen III and α-SMA and increased the expression of myocardial specific marker cTnT and gap junction protein Cx43 in CFs, enhanced cardiac reprogramming as opposed to 2 weeks with 5-aza alone. Transcriptome and quantitative real-time reverse transcription-polymerase chain reaction analysis at the 14th day postinduction of A83-01 revealed that the expression of genes involved in cardiac development increased in the presence of 5-aza. These findings suggest that the addition of A83-01 remarkably inhibits profibrotic signalling and improved the efficiency of iCMs, provide new insights into the molecular mechanisms of cardiac reprogramming and promote the use of iCMs in clinical applications.

Transcription factor Tbx5 promotes cardiomyogenic differentiation of cardiac fibroblasts treated with 5-azacytidine.

Conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes has recently been demonstrated, represents a potential therapeutic strategy for cardiac repair after myocardial injury. However, current approaches are inefficient. Here, we report that a defined transcription factor Tbx5, promoted cardiac reprogramming in the presence of a chemical inducer 5-azacytidine (5-aza). Morphological changes and cardiac specific genes and proteins expression were determined by immunofluorescence, quantitative real-time polymerase chain reaction, and Western blot analysis. Remarkably, Tbx5 enabled cardiac reprogramming with 5-aza by activating the expression of myocardial transcription-related genes, including cTnT, α-actin, Mef2c and inhibiting the expression of pluripotent genes such as Nanog, Oct4, and Sox2. Moreover, overexpression of Tbx5 upregulated the expression of sarcomere protein cTnT in CFs more efficiently at week 3 compared with 5aza-treated alone (P < 0.05). Conversely, inhibition of Tbx5 attenuated cardiac reprogramming. Furthermore, downregulated Tbx5 decreased wnt3a expression. At the same time, the inhibition effect of Tbx5i on cardiac reprogramming was reversed in vitro when these cells were exposed to Chir99021, a GSK-3 inhibitor. This finding provides new insight into the mechanism of cardiac reprogramming underlying the cardiac reprogramming process and lays the foundation for future clinical applications.

A new structure from cardiac cells cultured in vitro: Cardiomyocyte-annulation of neonatal rats.

To explore the formation, morphological characteristics, cell composition, and differentiation potential of cardiomyocyte annulation (cardio-annulation) during in vitro culture of cardiac cells. Cardiac cells were isolated and cultured. A live-cell imaging system was used to observe cardio-annulation. Cardiac troponin-T (cTnT) and vimentin were labeled with double immunofluorescence staining, and coexpressions of cTnT and connexin43 (Cx43), cTnT and nanog, c-kit and nanog, and c-kit and stem cell antigen (sca-1) were detected. The location of various types of cells within the cardio-annulation structure was observed. Adipogenic- and osteogenic-inducing fluids were used separately for in situ induction to detect the multidirectional differentiation potential of cells during the annulation process. After 3 to 6 days, cardiac cells migrated and formed an open or closed annulus with a diameter of 800 to 3500 µm. The annulus wall comprised the medial, middle, and lateral regions. The cells in the medial region were small, abundant, and laminated, while those in the middle region were larger with fewer layers, and those in the lateral region were less abundant, and loosely arranged in a single layer. Cardiomyocytes were distributed mainly on the surface of the medial region; nanog+ , c-kit+ , and sca-1+ cells were located mainly at the bottom of the annulus wall and fibroblasts were located mainly between these layers. The annulus cavity contained a large number of small, round cells, and telocytes. Cx43 was expressed in all cell types, and nanog, c-kit, and sca-1 were coexpressed in the cardio-annulation cells, which possess adipogenic and osteogenic differentiation potential. Cardio-annulation was discovered during an in vitro culture of cardiac cells. The structure contains cardiomyocytes, fibroblasts, telocytes, and abundant stem cells. These results provide insight into the relationship among cardiac cells in vitro.

CD90+ cardiac fibroblasts reduce fibrosis of acute myocardial injury in rats.

OBJECTIVE: To explore the differentiation tendency of CD90+ cardiac fibroblast (CFs) into cardiomyogenic cells in vitro and repair functions in acute myocardial infarction rats. METHODS: CD90+ subpopulation was sorted from rat CFs by flow cytometry. 10 µmoL/L of 5-Azacytosine (5-aza) was used to induce differentiation of CFs into cardiomyogenic cells. An acute myocardial infarction model was prepared by ligation of the rat left anterior descending coronary artery. After nuclei were labeled by DAPI, induced CD90+ CFs were injected into the infarction marginal zone. Before coronary ligation, 40 min after ligation, and at 7 and 14 days after cell transplantation, cardiac function changes were detected by ultrasound imaging system respectively. cTnT and endothelial cell marker VIII factor were detected by immunofluorescence staining. Infarct size was examined by TTC staining. Fibrosis was evaluated with masson's trichrome staining, vimentin, type I and type III collagen staining. RESULTS: CD90+ CFs sorted by flow cytometry was 34.9%. On day 28 after induction, the cTnT positive rate was 61.17 ±â€¯9.75%. Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) at days 7 and 14 after transplantation were significantly increased compared with those before transplantation (P < 0.05). LVEF and LVFS at the 14th day after transplantation were also significantly increased compared with those at the 7th day (LVEF: 61.40 ±â€¯2.45% vs. 56.25 ±â€¯2.9%, LVFS: 33.21 ±â€¯0.68% vs. 30.26 ±â€¯2.06%, P < 0.01). Additionally, small numbers of CD90+ CFs differentiated into cardiomyocytes and became involved in neovascularization. CD90+ CFs and CFs reduced myocardial infarct size at days 14. It was significantly smaller in rats with CFs transplantation group than those in MI group(24.78 ±â€¯2.28% vs. 31.28 ±â€¯2.83%, P < 0.05), and it was also significantly smaller in rats with CD90+CFs transplantation group than those in CFs transplantation group (17.47 ±â€¯4.15% vs. 24.78 ±â€¯2.28%, P < 0.05). Meanwhile, The percentage of fibrotic area and vimentin, type I and type III collagen in the infarct border zone and infarct area were both significantly reduced in CD90 + CFs. CONCLUSION: CD90+ CFs is the preponderant subpopulation of cardiomyogenic differentiation, with potential use as seed cells in basic and clinical research of heart regeneration and repair.

Multiple Directional Differentiation Difference of Neonatal Rat Fibroblasts from Six Organs.

BACKGROUND/AIMS: Fibroblasts are abundantly distributed throughout connective tissues in the body and are very important in maintaining the structural and functional integrity. Recent reports have proved that fibroblasts and mesenchymal stem cells share much more in common than previously recognized. The aim of this study was to investigate comparative studies in fibroblasts on the differences in the expression of molecular markers and differentiation capacity from different organs. METHODS: Combined trypsin/collagenase enzymes digestion method was used to isolate and culture the fibroblasts derived from heart, liver, spleen, lung, kidney and skin. Cell activity was determined by methyl thiazolyl tetrazolium (MTT) assay. Common molecular markers for fibroblasts such as vimentin, DDR2 and FSP1, stem cell markers nanog, c-kit and sca-1 were detected by RT-PCR, immunofluorescence and western blotting. The osteogenic, adipogenic and cardiogenic differentiations of fibroblasts were performed by inductive culture in special mediums, and analyzed by Alizarin red, Oil red O and immunofluorescence staining of cTnT respectively. RESULTS: The proliferation rate of fibroblasts in lung was faster than in other five organs. Common molecular markers for fibroblasts were expressed differently in different organs. DDR2 was strongly expressed in fibroblasts in the heart, partly expressed in the heart, skin, liver and spleen. Interestingly, no expression of DDR2 was detected in liver and kidney. However, vimentin and FSP1 were consistently expressed in fibroblasts from skin, liver, kidney, spleen and lung. nanog expression in fibroblasts from lung was less than that from heart, skin, liver and spleen (P < 0.01). c-kit expression in fibroblasts from heart, skin and kidney was higher than that from spleen (P < 0.05), while the c-kit positive fibroblasts from liver was obviously higher than that from spleen (P < 0.01). But sca-1 expression in fibroblasts from lung was the lowest among six organs (P < 0.01). Directed differentiation in vitro had demonstrated that skin fibroblasts had the strongest multiple differentiation potential, and the next was cardiac fibroblasts. And fibroblasts in liver and kidney had the advantage in myocardial differentiation, while fibroblasts in spleen only had the advantage in osteogenic differentiation. CONCLUSIONS: There are obvious heterogeneity in molecular markers and muti-directional differentiation in fibroblasts from six organs.

Telocytes in the Spleen.

Telocytes, a novel type of interstitial cells with very long and thin prolongations, have been identified in many organs in mammals. At present, the ultrastructural, immunocytochemical and electrophysiological properties of telocytes in multiple organs have been understood. However, telocytes in spleen, especially their roles in spleen have not been reported. The aim of this study was to investigate the ultrastructure, distribution and immunophenotypes of splenic telocytes. Rat spleen was harvested for the ultrastructure analysis by transmission electron microscopy (TEM). The primary culture of telocytes was performed after combined enzymatic digestion. The characteristic morphology was analyzed by a scanning electron microscopy (SEM). It was shown that telocytes displayed a piriform/spindle/triangular shape with long and slender telopods and extremely long prolongation contracting with surrounding cells in the spleen. Their dynamic profiles of cytoplasmic separation were recorded by the Live Cell Imaging System. The length of telopods was mostly distributing in 20-30 µm, in accordance with normal distribution. Most telocytes had three or two telopods (28.71% and 22.58% respectively). Immunostaining indicated that these cells were positive for vimentin, CD34, nanog and sca-1, but negative for c-kit. These data prove the existence of telocytes in the spleen, which may serve as the experimental base for exploring their roles in the spleen.

Multiple immunophenotypes of cardiac telocytes.

AIMS: Telocytes (TCs) form a 3-dimensional network in the myocardial interstitium, which most probably play important role(s) in heart development. However, the dynamics of their prolongations, continuous cell shape changes and adherence properties have not been well documented till recently. The aim of this study was to investigate dynamics of extension of prolongations (Telopods) and multiple phenotypes of cardiac TCs cultured in vitro. METHODS: Cardiac TCs were isolated from neonatal rats by a combined enzyme digestion process and identified by light microscopy, immunofluorescence analysis and scanning using electron microscopy (SEM). Their continuous changes in shape were analyzed by a Live Cell Imaging System and multiple phenotypes were identified by immunofluorescence analysis using various markers, like vimentin, c-kit, CD34, nanog and sca-1. RESULTS: Cardiac TCs displayed piriform/spindle/triangular shapes with long and slender telopodes showing extremely long prolongations. The morphology of cell body was continuously changing while their prolongations were extending gradually. After adhering to the surface, TCs' movement and extension of their prolongations lasted for approximately 1.5h. Cardiac TCs expressed mesenchymal cell marker vimentin, hematopoietic stem cell marker CD34, embryonic stem cell-associated gene of Nanog, and myocardial stem cell markers sca-1 and c-kit. CONCLUSION: These findings indicate that cultured TCs in vitro have multiple phenotypes, which are most likely important for evaluating their functional roles in heart development.

Gata4, Tbx5 and Baf60c induce differentiation of adipose tissue-derived mesenchymal stem cells into beating cardiomyocytes.

The adipose tissue-derived mesenchymal stem cells (ADMSCs) are extensively utilized in tissue engineering, regenerative medicine and cell therapy. ADMSCs can differentiate into cardiomyocytes, and it has been shown that over-expression of a cocktail of factors can induce ectopic heart formation and program cardiogenesis in ESCs. However, which genes are responsible for differentiation of ADMSCs into beating cardiomyocyte-like cells remains unknown. In this study we have shown that the combination of Gata4, Tbx5 and Baf60c is sufficient for inducing ADMSCs to form cardiomyocytes. It also appears that, while Gata4 and Baf60c are key inducers of myocardial differentiation, Tbx5 is essential for the ability of cardiac cells to contract. These findings provide additional experimental references for myocardial tissue engineering in the emerging field of cell-based therapy of heart diseases.

The expression analysis of nanog in the developing rat myocardial tissues.

OBJECTIVES: To investigate the expression dynamic of nanog gene in the development of rat myocardial tissues. METHODS: SD rats were studied at 5 time points before and after birth. The techniques of immunohistochemistry, immunofluorescence, western blotting and RT-PCR were used to investigate the expression of nanog gene in the rat myocardial tissues at different embryonic (E) and postnatal (P) stages, and image analysis system was used for the quantitative analysis. RESULTS: The immunohistochemistry, immunofluorescence and western blotting analyses have shown that expression of nanog protein was highest in the rat myocardial tissues at E18, then it gradually declined at postnatal stages (P<0.05), and became nearly undetectable in most myocardial tissues at P30 with very few remaining nanog-positive cells. RT-PCR result indicated that the expression of nanog gene was strong at E18, but gradually decreased from E18 to P30. CONCLUSION: The mRNA transcription and protein translation of nanog gene in the rat heart gradually decreased with every consecutive growth stage. This indicates that nanog gene has potential regulatory functions in the differentiation of myocardial cells during rat development.

Mesenchymal stem cell-like properties in fibroblasts.

Fibroblasts are biologically dynamic and morphologically heterogeneous and are the most abundant connective tissue cells, with diverse structures depending on their location and activity. The main function of fibroblasts is to maintain the structural integrity of connective tissues by continuously secreting precursors of the extracellular matrix. Recent advances in our knowledge on pathophysiologic features of fibroblasts revealed that in some situations epithelial cells can give rise to fibroblasts by epithelial-mesenchymal transition (EMT) and conversely, in some other situations, fibroblasts may give rise to epithelia by undergoing a mesenchymal to epithelial transition (MET). Given an opportunity to differentiate to other cells, fibroblasts may foster a novel clue for in situ tissue repair and contribute to cellular mechanisms of mesenchymal stem cell-like features under normal or pathological conditions. They have also been shown to suppress immune responses in vitro. Because of these properties, fibroblasts have recently received a very high profile in the literature. This review summarizes our understanding of the origins, mesenchymal stem cell-like characteristics and potency of directed differentiation of fibroblasts. In addition, we also present the evidence that mesenchymal stem cells and fibroblasts share much more in common than previously recognized.