Yasheng Yan

IFATS Collection: Human Adipose-Derived Stem Cells Seeded on a Silk Fibroin-Chitosan Scaffold Enhance Wound Repair in a Murine Soft Tissue Injury Model†‡§

Soft tissue loss presents an ongoing challenge in reconstructive surgery. Local stem cell application has recently been suggested as a possible novel therapy. In the present study we evaluated the potential of a silk fibroin‐chitosan (SFCS) scaffold serving as a delivery vehicle for human adipose‐derived stem cells (ASCs) in a murine soft tissue injury model. Green fluorescent protein (GFP)‐labeled ASCs were seeded on SFCS scaffolds at a density of 1 × 105 ASCs per cm2 for 48 hours and then suture‐inlaid to a 6‐mm, full‐thickness skin defect in 6‐week‐old male athymic mice. Wound healing was tracked for 2 weeks by planimetry. Histology was evaluated at 2 and 4 weeks. Our data show that the extent of wound closure was significantly enhanced in the ASC‐SFCS group versus SFCS and no‐graft controls at postoperative day 8 (90% ± 3% closure vs. 75% ± 11% and 55% ± 17%, respectively). Microvessel density at wound bed biopsy sites from 2 weeks postoperative was significantly higher in the ASC‐SFCS group versus SFCS alone (7.5 ± 1.1 vs. 5.1 ± 1.0 vessels per high‐power field). Engrafted stem cells were positive for the fibroblastic marker heat shock protein 47, smooth muscle actin, and von Willebrand factor at both 2 and 4 weeks. GFP‐positive stem cells were also found to differentiate into epidermal epithelial cells at 4 weeks postoperative. In conclusion, human adipose‐derived stem cells seeded on a silk fibroin‐chitosan scaffold enhance wound healing and show differentiation into fibrovascular, endothelial, and epithelial components of restored tissue. STEM CELLS 2009;27:250–258

Both cultured and freshly isolated adipose tissue-derived stem cells enhance cardiac function after acute myocardial infarction

AIMS We assessed whether freshly isolated human adipose tissue-derived cells (fhADCs) or cultured human adipose tissue-derived stem cells (hASCs) have beneficial effects on cardiac function after myocardial infarction (MI), whether the injected cells can survive long term, and whether their effects result from direct differentiation or paracrine mechanisms. METHODS AND RESULTS Myocardial infarction was experimentally induced in severe combined immunodeficient mice, and either fhADCs, cultured hASCs, or phosphate-buffered saline was injected into the peri-infarct region. Myocardial function improved significantly in mice treated with hASCs or fhADCs 4 weeks after MI. Immunofluorescence revealed that grafted hASCs and fhADCs underwent cardiomyogenic differentiation pathway, as indicated by expression of connexin 43 and troponin I in a fusion-independent manner. Some of the injected cells integrated with host cardiomyocytes through connexin 43, and others were incorporated into newly formed vessels. Human adipose tissue-derived stem cells survived in injured hearts up to 4 months, as detected by luciferase-based bioluminescence imaging. Vascular density was significantly increased, and fewer apoptotic cells were present in the peri-infarct region of cell-injected mice. CONCLUSION This is the first study to systematically compare the effects of fhADCs and hASCs on myocardial regeneration. Both cell types engraft into infarcted myocardium, survive, and improve myocardial function, suggesting that fhADCs, like hASCs, are a promising alternative cell source for myocardial repair after MI.

Fibroblasts share mesenchymal phenotypes with stem cells, but lack their differentiation and colony-forming potential

Background information. Although MSCs (mesenchymal stem cells) and fibroblasts have been well studied, differences between these two cell types are not fully understood. We therefore comparatively analysed antigen and gene profiles, colony‐forming ability and differentiation potential of four human cell types in vitro: commercially available skin‐derived fibroblasts [hSDFs (human skin‐derived fibroblasts)], adipose tissue‐derived stem cells [hASCs (human adipose tissue‐derived stem cells)], embryonic lung fibroblasts (WI38) and dermal microvascular endothelial cells [hECs (human dermal microvascular endothelial cells)].

Dermal matrix as a carrier for in vivo delivery of human adipose-derived stem cells

The aim of the present study was to evaluate the potential of acellular dermal matrix as a carrier for delivery of stem cells to the site of soft tissue defect in a murine skin injury model and to determine the potential of stem cells delivered via such an approach to successfully engraft, survive and differentiate locally. We showed that adipose-derived stem cells delivered via this matrix survived after in vivo engraftment, spontaneously differentiated along vascular endothelial, fibroblastic and epidermal epithelial lineages and significantly improved wound healing. Furthermore, an organ survey for transplanted cells showed no evidence of a systemic distribution beyond the cutaneous wound site, indicating that the adipose-derived stem cell-dermal matrix construct provides a novel and effective method for anatomically focused cellular therapy. In conclusion, stem cell-seeded dermal matrix is an effective means for targeted in vivo cell delivery for enhanced soft tissue regeneration.

Ketamine enhances human neural stem cell proliferation and induces neuronal apoptosis via reactive oxygen species-mediated mitochondrial pathway.

BACKGROUND:Growing evidence indicates that ketamine causes neurotoxicity in a variety of developing animal models, leading to a serious concern regarding the safety of pediatric anesthesia. However, if and how ketamine induces human neural cell toxicity is unknown. Recapitulation of neurogenesis from human embryonic stem cells (hESCs) in vitro allows investigation of the toxic effects of ketamine on neural stem cells (NSCs) and developing neurons, which is impossible to perform in humans. In the present study, we assessed the influence of ketamine on the hESC-derived NSCs and neurons. METHODS:hESCs were directly differentiated into neurons via NSCs. NSCs and 2-week-old neurons were treated with varying doses of ketamine for different durations. NSC proliferation capacity was analyzed by Ki67 immunofluorescence staining and bromodeoxyuridine assay. Neuroapoptosis was analyzed by TUNEL staining and caspase 3 activity measurement. The mitochondria-related neuronal apoptosis pathway including mitochondrial membrane potential, cytochrome c distribution within cells, mitochondrial fission, and reactive oxygen species (ROS) production were also investigated. RESULTS:Ketamine (100 µM) increased NSC proliferation after 6-hour exposure. However, significant neuronal apoptosis was only observed after 24 hours of ketamine treatment. In addition, ketamine decreased mitochondrial membrane potential and increased cytochrome c release from mitochondria into cytosol. Ketamine also enhanced mitochondrial fission as well as ROS production compared with no-treatment control. Importantly, Trolox, a ROS scavenger, significantly attenuated the increase of ketamine-induced ROS production and neuronal apoptosis. CONCLUSIONS:These data for the first time demonstrate that (1) ketamine increases NSC proliferation and causes neuronal apoptosis; (2) mitochondria are involved in ketamine-induced neuronal toxicity, which can be prevented by Trolox; and (3) the stem cell–associated neurogenesis system may provide a simple and promising in vitro model for rapidly screening anesthetic neurotoxicity and studying the underlying mechanisms as well as prevention strategies to avoid this toxic effect.

Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death

Myocardial ischemia-reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1 (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of mitochondrial fission; and (2) the increased mitochondrial fission is resulted from both increased activation and decreased inactivation of Drp1 through Cdk1, PKCδ, and calcineurin-mediated pathways, respectively.

Ketamine induces toxicity in human neurons differentiated from embryonic stem cells via mitochondrial apoptosis pathway

Ketamine is widely used for anesthesia in pediatric patients. Growing evidence indicates that ketamine causes neurotoxicity in a variety of developing animal models. Our understanding of anesthesia neurotoxicity in humans is currently limited by difficulties in obtaining neurons and performing developmental toxicity studies in fetal and pediatric populations. It may be possible to overcome these challenges by obtaining neurons from human embryonic stem cells (hESCs) in vitro. hESCs are able to replicate indefinitely and differentiate into every cell type. In this study, we investigated the toxic effect of ketamine on neurons differentiated from hESCs. Two-week-old neurons were treated with different doses and durations of ketamine with or without the reactive oxygen species (ROS) scavenger, Trolox. Cell viability, ultrastructure, mitochondrial membrane potential (ΔΨm), cytochrome c distribution within cells, apoptosis, and ROS production were evaluated. Here we show that ketamine induced ultrastructural abnormalities and dose- and time-dependently caused cell death. In addition, ketamine decreased ΔΨm and increased cytochrome c release from mitochondria. Ketamine also increased ROS production and induced differential expression of oxidative stress-related genes. Specifically, abnormal ultrastructural and ΔΨm changes occurred earlier than cell death in the ketamine-induced toxicity process. Furthermore, Trolox significantly decreased ROS generation and attenuated cell death caused by ketamine in a dose-dependent manner. In conclusion, this study illustrates that ketamine time- and dose-dependently induces human neurotoxicity at supraclinical concentrations via ROS-mediated mitochondrial apoptosis pathway and that these side effects can be prevented by the antioxidant agent Trolox. Thus, hESC-derived neurons might provide a promising tool for studying anesthetic-induced developmental neurotoxicity and prevention strategies.

Down-regulation of microRNA-21 is involved in the propofol-induced neurotoxicity observed in human stem cell-derived neurons.

Background:Recent studies in various animal models have suggested that anesthetics such as propofol, when administered early in life, can lead to neurotoxicity. These studies have raised significant safety concerns regarding the use of anesthetics in the pediatric population and highlight the need for a better model to study anesthetic-induced neurotoxicity in humans. Human embryonic stem cells are capable of differentiating into any cell type and represent a promising model to study mechanisms governing anesthetic-induced neurotoxicity. Methods:Cell death in human embryonic stem cell–derived neurons was assessed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling staining, and microRNA expression was assessed using quantitative reverse transcription polymerase chain reaction. miR-21 was overexpressed and knocked down using an miR-21 mimic and antagomir, respectively. Sprouty 2 was knocked down using a small interfering RNA, and the expression of the miR-21 targets of interest was assessed by Western blot. Results:Propofol dose and exposure time dependently induced significant cell death (n = 3) in the neurons and down-regulated several microRNAs, including miR-21. Overexpression of miR-21 and knockdown of Sprouty 2 attenuated the increase in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling–positive cells following propofol exposure. In addition, miR-21 knockdown increased the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling–positive cells by 30% (n = 5). Finally, activated signal transducer and activator of transcription 3 and protein kinase B (Akt) were down-regulated, and Sprouty 2 was up-regulated following propofol exposure (n = 3). Conclusions:These data suggest that (1) human embryonic stem cell–derived neurons represent a promising in vitro human model for studying anesthetic-induced neurotoxicity, (2) propofol induces cell death in human embryonic stem cell–derived neurons, and (3) the propofol-induced cell death may occur via a signal transducer and activator of transcription 3/miR-21/Sprouty 2–dependent mechanism.

Human adipose tissue-derived stem cells exhibit proliferation potential and spontaneous rhythmic contraction after fusion with neonatal rat cardiomyocytes

Various types of stem cells have been shown to have beneficial effects on cardiac function. It is still debated whether fusion of injected stem cells with local resident cardiomyocytes is one of the mechanisms. To better understand the role of fusion in stem cell‐based myocardial regeneration, the present study was designed to investigate the fate of human adipose tissue‐derived stem cells (hASCs) fused with neonatal rat cardiomyocytes in vitro. hASCs labeled with the green fluorescent probe Vybrant DiO were cocultured with neonatal rat cardiomyocytes labeled with the red fluorescent probe Vybrant Dil and then treated with fusion‐inducing hemagglutinating virus of Japan (HVJ). Cells that incorporated both red and green fluorescent signals were considered to be hASCs that had fused with rat cardiomyocytes. Fusion efficiency was 19.86 ± 4.84% at 5 d after treatment with HVJ. Most fused cells displayed cardiomyocyte‐like morphology and exhibited spontaneous rhythmic contraction. Both immunofluorescence staining and lentiviral vector labeling showed that fused cells contained separate rat cardiomyocyte and hASC nuclei. Immunofluorescence staining assays demonstrated that human nuclei in fused cells still expressed the proliferation marker Ki67. In addition, hASCs fused with rat cardiomyocytes were positive for troponin I. Whole‐cell voltage‐clamp analysis demonstrated action potentials in beating fused cells. RT‐PCR analysis using rat‐ or human‐specific myosin heavy chain primers revealed that the myosin heavy‐chain expression in fused cells was derived from rat cardiomyocytes. Real‐time PCR identified expression of human troponin T in fused cells and the presence of rat cardiomyocytes induced a cardiomyogenic protein expression of troponin T in human ASCs. This study illustrates that hASCs exhibit both stem cell (proliferation) and cardiomyocyte properties (action potential and spontaneous rhythmic beating) after fusion with rat cardiomyocytes, supporting the theory that fusion, even if artificially induced in our study, could indeed be a mechanism for cardiomyocyte renewal in the heart.—Metzele, R., Alt, C., Bai, X., Yan, Y., Zhang, Z., Pan, Z., Coleman, M., Vykoukal, J., Song, Y.‐H., Alt, E. Human adipose tissue‐derived stem cells exhibit proliferation potential and spontaneous rhythmic contraction after fusion with neonatal rat cardiomyocytes. FASEB J. 25, 830–839 (2011).

MicroRNA-21 Mediates Isoflurane-induced Cardioprotection against Ischemia-Reperfusion Injury via Akt/Nitric Oxide Synthase/Mitochondrial Permeability Transition Pore Pathway.

Background:The role of microRNA-21 in isoflurane-induced cardioprotection is unknown. The authors addressed this issue by using microRNA-21 knockout mice and explored the underlying mechanisms. Methods:C57BL/6 and microRNA-21 knockout mice were echocardiographically examined. Mouse hearts underwent 30 min of ischemia followed by 2 h of reperfusion in vivo or ex vivo in the presence or absence of 1.0 minimum alveolar concentration of isoflurane administered before ischemia. Cardiac Akt, endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) proteins were determined by Western blot analysis. Opening of the mitochondrial permeability transition pore (mPTP) in cardiomyocytes was induced by photoexcitation-generated oxidative stress and detected by rapid dissipation of tetramethylrhodamine ethyl ester fluorescence using a confocal microscope. Results:Genetic disruption of miR-21 gene did not alter phenotype of the left ventricle, baseline cardiac function, area at risk, and the ratios of phosphorylated-Akt/Akt, phosphorylated-eNOS/eNOS, and phosphorylated-nNOS/nNOS. Isoflurane decreased infarct size from 54 ± 10% in control to 36 ± 10% (P < 0.05, n = 8 mice per group), improved cardiac function after reperfusion, and increased the ratios of phosphorylated-Akt/AKT, phosphorylated-eNOS/eNOS, and phosphorylated-nNOS/nNOS in C57BL/6 mice subjected to ischemia–reperfusion injury. These beneficial effects of isoflurane were lost in microRNA-21 knockout mice. There were no significant differences in time of the mPTP opening induced by photoexcitation-generated oxidative stress in cardiomyocytes isolated between C57BL/6 and microRNA-21 knockout mice. Isoflurane significantly delayed mPTP opening in cardiomyocytes from C57BL/6 but not from microRNA-21 knockout mice. Conclusions:Isoflurane protects mouse hearts from ischemia–reperfusion injury by a microRNA-21-dependent mechanism. The Akt/NOS/mPTP pathway is involved in the microRNA-21-mediated protective effect of isoflurane.