Deciphering molecular heterogeneity and dynamics of human hippocampal neural stem cells at different ages and injury states.

While accumulated publications support the existence of neurogenesis in the adult human hippocampus, the homeostasis and developmental potentials of neural stem cells (NSCs) under different contexts remain unclear. Based on our generated single-nucleus atlas of the human hippocampus across neonatal, adult, aging, and injury, we dissected the molecular heterogeneity and transcriptional dynamics of human hippocampal NSCs under different contexts. We further identified new specific neurogenic lineage markers that overcome the lack of specificity found in some well-known markers. Based on developmental trajectory and molecular signatures, we found that a subset of NSCs exhibit quiescent properties after birth, and most NSCs become deep quiescence during aging. Furthermore, certain deep quiescent NSCs are reactivated following stroke injury. Together, our findings provide valuable insights into the development, aging, and reactivation of the human hippocampal NSCs, and help to explain why adult hippocampal neurogenesis is infrequently observed in humans.

PLGA-PNIPAM Microspheres Loaded with the Gastrointestinal Nutrient NaB Ameliorate Cardiac Dysfunction by Activating Sirt3 in Acute Myocardial Infarction.

Acute myocardial infarction (AMI) is the death of cardiomyocytes caused by a lack of energy due to ischemia. Nutrients supplied by the blood are the main source of cellular energy for cardiomyocytes. Sodium butyrate (NaB), a gastrointestinal nutrient, is a short-chain fatty acid (butyric acid) that may act as an energy source in AMI therapy. Poly(lactic-co-glycolic acid)-Poly (N-isopropylacrylamide) microspheres loaded with NaB (PP-N) are synthesized to prolong the release of NaB and are injected into ischemic zones in a Sprague-Dawley rat AMI model. Here, this study shows that PP-N can significantly ameliorate cardiac dysfunction in AMI, and NaB can specially bind to Sirt3 structure, activating its deacetylation ability and inhibiting the generation of reactive oxygen species, autophagy, and angiogenesis promotion. The results indicate that NaB, acting as a nutrient, can protect cardiomyocytes in AMI. These results suggest that the gastrointestinal nutrient NaB may be a new therapy for AMI treatment, and PP-N may be the ideal therapeutic regimen.

Construction of an Aptamer-SiRNA Chimera-Modified Tissue-Engineered Blood Vessel for Cell-Type-Specific Capture and Delivery.

The application of tissue-engineered blood vessels (TEBVs) is the main developmental direction of vascular replacement therapy. Due to few and/or dysfunctional endothelial progenitor cells (EPCs), it is difficult to successfully construct EPC capture TEBVs in diabetes. RNA has a potential application in cell protection and diabetes treatment, but poor specificity and low efficiency of RNA transfection in vivo limit the application of RNA. On the basis of an acellular vascular matrix, we propose an aptamer-siRNA chimera-modified TEBV that can maintain a satisfactory patency in diabetes. This TEBV consists of two parts, CD133-adenosine kinase (ADK) chimeras and a TEBV scaffold. Our results showed that CD133-ADK chimeras could selectively capture the CD133-positive cells in vivo, and then captured cells can internalize the bound chimeras to achieve RNA self-transfection. Subsequently, CD133-ADK chimeras were cut into ADK siRNA by a dicer, resulting in depletion of ADK. An ADK-deficient cell may act as a bioreactor that sustainably releases adenosine. To reduce nonspecific RNA transfection, we increased the proportion of HAuCl4 during the material preparation, through which the transfection capacity of polyethylenimine (PEI)/polyethylene glycol (PEG)-capped gold nanoparticles (PEI/PEG-AuNPs) was significantly decreased and the ability of TEBV to resist tensile and liquid shear stress was greatly enhanced. PEG and 2'-O-methyl modification was used to enhance the in vivo stability of RNA chimeras. At day 30 postgrafting, the patency rate of CD133-ADK chimera-modified TEBVs reached 90% in diabetic rats and good endothelialization was observed.

Regulation of Cellular Response Pattern to Phosphorus Ion is a New Target for the Design of Tissue-Engineered Blood Vessel.

Regulation of cellular response pattern to phosphorus ion (PI) is a new target for the design of tissue-engineered materials. Changing cellular response pattern to high PI can maintain monocyte/macrophage survival in TEBV and the signal of increasing PI can be converted by klotho to the adenosine signals through the regulation of energy metabolism in monocytes/macrophages.

The use of BDNF to enhance the patency rate of small-diameter tissue-engineered blood vessels through stem cell homing mechanisms.

The patency rate of small-diameter tissue-engineered blood vessels is the determinant for their application in coronary artery bypass grafting. The coronary artery is innervated by vagus nerves. The origin of vagus nerves is rich in brain-derived neurotrophic factors (BDNF). We have investigated whether BDNF could improve the patency rate of small-diameter tissue-engineered blood vessels through promoting stem cell homing and paracrine activity. In vitro, we isolated early and late endothelial progenitor cells (EPCs) and found BDNF could promote single clone formation and paracrine function of EPCs, and could also induce the proliferation, migration and differentiation of late EPCs. BDNF could enhance the capturing of EPCs in parallel-plate flow chamber. Flow cytometric analysis and laser-scanning confocal microscope showed BDNF could enhance the mobilization and homing of C57BL/6 mouse EPCs after wire injury. Based on it, BDNF was coupled to the lumen surface of the blood vessel matrix material incubated with collagen through SPDP to construct BDNF-modified small-diameter tissue-engineered blood vessel. The blood vessel patency rate was significantly higher than that of control group 8 weeks after implantation in rats and the endothelialization level was superior to control. These results demonstrate that BDNF can effectively improve patency of small-diameter tissue-engineered blood vessels through stem cell homing and paracrine, and it is expected to play an important role in the construction of substitutes for coronary artery bypass grafting.

The promotion of endothelial progenitor cells recruitment by nerve growth factors in tissue-engineered blood vessels.

Endothelial progenitor cells (EPCs) mobilization and homing are critical to the development of an anti-thrombosis and anti-stenosis tissue-engineered blood vessel. The growth and activation of blood vessels are supported by nerves. We investigated whether nerve growth factors (NGF) can promote EPCs mobilization and endothelialization of tissue-engineered blood vessels. In vitro, NGF promoted EPCs to form more colonies, stimulated human EPCs to differentiate into endothelial cells, and significantly enhanced EPCs migration. Flow cytometric analysis revealed that NGF treatment increased the number of EPCs in the peripheral circulation of C57BL/6 mice. Furthermore, the treatment of human EPCs with NGF facilitated their homing into wire-injured carotid arteries after injection into mice. Decellularized rat blood vessel matrix was incubated with EDC cross-linked collagen and bound to NGF protein using the bifunctional coupling agent N-succinmidyl3-(2-pyridyldit-hio) propionate (SPDP). The NGF-bound tissue-engineered blood vessel was implanted into rat carotid artery for 1 week and 1 month. NGF-bound blood vessels possessed significantly higher levels of endothelialization and patency than controls did. These results demonstrated that NGF can markedly increase EPCs mobilization and homing to vascular grafts. Neurotrophic factors such as NGF have a therapeutic potential for the construction of tissue-engineered blood vessels in vivo.

Bone regeneration using an acellular extracellular matrix and bone marrow mesenchymal stem cells expressing Cbfa1.

To treat bone defects, tissue-engineering methods combine an appropriate scaffold with cells and osteogenic signals to stimulate bone repair. Mesenchymal stem cells (MSCs) derived from adult bone marrow are an ideal source of cells for tissue engineering, in particular for applications in skeletal and hard tissue repair. Core binding factor alpha1 (Cbfa1) is an essential transcription factor for osteoblast differentiation. However, the effects of Cbfa1 on MSCs in vitro and in vivo have not been well characterized. In this study, we found that MSCs modified genetically to express Cbfa1 promoted the healing of segmental defects of the radius in rabbits. First, osteogenic differentiation of MSCs transfected with an adenovirus encoding Cbfa1 was demonstrated. Expression of mRNA from a number of osteoblastic marker genes, including osteocalcin, osteopontin, and type I collagen, was detected. In addition, alkaline phosphatase activity and increased osteocalcin content were observed. The cells expressing the Cbfa1 gene were then combined with acellular bone extracellular matrix in a flow perfusion culture system. Finally, the cell-matrix constructs were implanted into radius defects in the rabbit model. After 12 weeks, radiographic, histological, and biomechanical analyses showed that MSCs modified with the Cbfa1 gene resulted in a significantly higher amount of newly-formed bone and rebuilding of the marrow cavity than control cell-matrix constructs. This study indicates that MSCs modified with the Cbfa1 gene can act as suitable seed cells for the regeneration of bone defects.

A20 overexpression inhibits low shear flow-induced CD14-positive monocyte recruitment to endothelial cells.

OBJECTIVE: To determine if A20, a zinc finger protein that mediates the inflammatory response, affects monocyte-endothelial cell-cell interactions induced by low shear flow. METHODS: Primary cultured endothelial cells (EC) were transfected with an A20 expression vector, and the VCAM-1, ICAM-1 and IL-8 mRNA, and protein expression levels in A20-transfected EC lysates were checked by PCR array and ELISA, respectively. CD14-positive monocyte migration toward and adhesion to EC were measured using a parallel plate flow chamber. RESULTS: Low shear stress, defined as 0.2 Pa for 6 h, normally increases VCAM-1, ICAM-1 and IL-8 expression in EC and allows for binding of monocytes to EC. We found that overexpression of A20 in EC inhibits their normal expression of VCAM-1, ICAM-1 and IL-8 under low shear stress conditions. We also found that A20 inhibits IkappaBalpha degradation in EC following low shear stress exposure and that it attenuates the rolling and EC adhesive properties of shear-induced monocytes as compared with untransfected control EC. The results demonstrate that A20 overexpression in EC inhibits low shear flow-induced monocyte-EC interactions. These findings may be useful in the development of therapeutic agents aimed at treating chronic inflammatory diseases like atherosclerosis.

Stereoscopic study on capillary density of early brain oedema in a dog postburn model.

BACKGROUND: After severe burn, the effective circulating blood volume decreases drastically due to massive body fluid loss, and blood redistribution occurs to maintain sufficient blood supply to vital organs. Blood perfusion of brain tissue changes and the permeability of the blood brain barrier increases due to ischaemia and hypoxia, which results in brain oedema. The goal of this study was to explore the changes of cerebral blood flow during the brain oedema at the early stage of severe burn. METHODS: Twenty-six dogs were randomly divided into control and 6, 12, 18, and 24 PBH groups. The manifestation of MRI and histopathology, changes of brain water content were investigated; the shapes and distribution of the cerebral capillaries were observed with the endogenous AKP histochemical staining method and image analysis technique. The volume, surface, and length fractions of cerebral capillaries were tested and analysed with a stereographic method in each group, respectively. RESULTS: The earliest changes of cerebral oedema were found at 12 PBH with MRI, which showed the brain swelling as characteristic of cerebral morphological changes. The decrease of SIR on T(1)WI was not observed until it was above 10%. Signal of T(2)WI increased for 8.29% at 24 PBH. Histological changes were observed as early as 6h after burn, accompanied by swelling of endothelial cells and peri-vascular astrocytes, vacuolation took place in neurons at 12h after burn, necrosis of different degrees in capillary endothelium, neurons, and axons. Increase in cerebral water content was noted at 6h postburn, and it was the most marked in 24 postburn group.The distributional density of capillaries became thicker at 6h and 12h postburn, the shapes were normal. The capillaries became sparser at 18h, and almost disappeared from view, only a few ends of capillaries in the shape of vine were seen at 24h postburn. The percentages of capillary volume, surface, and length fractions were increased at 6h and 12h, but decreased at 18h and reached the lowest point at 24h postburn (P<0.05). CONCLUSION: We suggest that the changes of cerebral blood flow might play an important role in the pathogenesis of brain oedema in the early stage of severe burn.

Development of anti-atherosclerotic tissue-engineered blood vessel by A20-regulated endothelial progenitor cells seeding decellularized vascular matrix.

To investigate whether decellularized vascular tissues and A20-regulated endothelial progenitor cells can be used for constructing a transgenic tissue-engineered blood vessel with anti-atherosclerotic vascular stenotic properties. A20 gene-transfected endothelial progenitor cells differentiated endothelial cells and smooth muscle cells attached to and migrated into the decellularized porcine vascular scaffolding in a bioreactor. The histology of the conduits revealed viable and layered tissue. Scanning electron microscopy showed confluent, homogeneous tissue surfaces. The mechanical strength of the pulsed constructs was similar to that of the human artery. In vivo, the A20 gene-transfected tissue-engineered blood vessels were transplanted into the carotid artery of a rat for 6 months. Blood vessel xenotransplantation caused hyperacute rejection; all transplanted control blood vessels were completely rejected, but A20-transfected tissue-engineered blood vessels demonstrated good flow on implantation, and remained open for 6 months postoperatively, as assessed by Doppler. The HE stain demonstrated that the vessels were patent, without evidence of stenosis or dilatation after 6 months. These results demonstrate that transgenic tissue-engineered blood vessels have long-term patency and unique anti-stenotic properties.

[Antigen measurement and biomechanical characteristics of the inbred-line Banna mini-pig acellular bone matrix].

In this study, we prepared the acellular bone matrix of the inbred-line Banna mini-pig by using tissue engineering method and evaluated its possible application in bone tissue engineering. Histological analysis, xenoantigen expression and biomechanical measurement were performed on the matrix. HE staining and scanning electron microscopy showed the cellular components were almost removed. Immunohischemical result demonstrated that the xenoantigen, alpha-gal,was also eliminated. There was no statistically significant difference between the acellular bone matrix group and control group. The acellular bone matrix can provide appropriate space structure and strength for grafts. In conclusion, our data suggest that acellular bone matrix is a new kind of ideal bone scaffold material.

Low shear stress regulates monocyte adhesion to oxidized lipid-induced endothelial cells via an IkappaBalpha dependent pathway.

In regions of a vessel that experience low shear stress and reversing flow patterns, early features in the pathogenesis of atherosclerosis include the accumulation of oxidized LDL (OxLDL) and adhesion of monocytes to endothelial cells (EC). Here we investigated the hypothesis that low shear stress (2 dyn/cm2) and OxLDL are synergistic for enhanced expression of vascular cell adhesion molecule (VCAM-1) and human aortic endothelial cell (HAEC)-monocyte adhesion. This study shows low shear stress can significantly reduce IkappaBalpha levels, activate NF-kappaB, increase the expression of VCAM-1 in HAEC and binding of monocytes. OxLDL itself cannot significantly increase the expression of VCAM-1 in HAEC and binding of monocytes, but through activation of NF-kappaB and degradation of IkappaBalpha induced by low shear stress it can significantly enhance VCAM-1 expression and monocyte adhesion, over that in unmodified LDL or control. These results suggest that low shear stress can regulate monocyte adhesion to oxidized lipid-induced endothelial cells via an IkappaBalpha-dependent pathway, and that low shear stress together with OxLDL may likely play an important role in atherogenesis.

[Antigen expression and the endothelization of biologic blood vessel matrix].

This study sought to explore the change of the major histocompatibility complex (MHC) antigen expression and the endothelization of blood vessel in minor pig after trypsin treatment, and to provide data for xenotransplantation and pig vessel for use in tissue engineering. Western blot assays were conducted for detecting the expression of MHC xenoantigens. Scanning electron microscopy was used for checking the endothelization of decellularized blood vessel. The results showed that MHC antigen is not expressed after trypsin treatment. The endothelization is accomplished. The endothelial cells have normal morphological distribution. These demonstrate that the antigen of pig aorta is significantly decreased and it can be used for constructing new vascular grafts.

The zinc finger protein A20 protects endothelial cells from burns serum injury.

Burn injuries as well as skin damages are often associated with immune suppression and often cause multiple organ failures. The monolayer endothelium is vulnerable to injuries from circulating factors resulting from remote wounds. Endothelial cell activation and apoptosis can alter microvascular permeability and intensify organ damage. A20, as a physiological cytoprotective gene is essential for preventing spontaneous innate immune cell-mediated inflammation and tissue destruction. It is not known whether A20 has the function to protect endothelial cells from the effect of burns serum challenge on endothelial function in vitro. This study shows that A20 can express in endothelial cells after burns serum stimulation and inhibit endothelial cell activation and apoptosis induced by burns serum. These results suggest that A20 may be beneficial in limiting the response to burn injuries.

[Anatomy of heart in banna mini-pig inbred-lines].

OBJECTIVE: To observe the heart anatomic and histological structure of the Banna mini-pig inbred-lined and to provide the morphological data for heart xenotransplantation and breeding transgens pig. METHODS: Ten Banna mini-pigs (12-18 months old) were affused and fixed by common coratid artery. The heart were observed and measured by gross anatomy and histology. RESULTS: There were many similarities between the Banna pig heart and the human heart in anatomy and histology. However, the following differences were observed in the Banna pig heart: 1. Azygos vein directly drew into right atrium cordis. 2. The intercalated disk of cardiac muscle was less than that of human. 3. The Purkinje's fibre was bigger than that of human. CONCLUSION: On the morphology and histology, the structure of Banna pig heart is similar to the heart of human being. It is possible that Banna minipig heart becomes organ donors for xenotransplantation.

[Protective effect of exogenous bcl-2 on spinal cord motoneurons following sciatic nerve axotomy in the rat].

The aim of this study was to investigate the protective effect of exogenous bcl-2 on spinal cord motoneurons following sciatic nerve axotomy. After epineurium suturing, sense bcl-2 (Ad/s-bcl-2), antisense bcl-2 (Ad/as-bcl-2), or reporter gene lacZ (Ad/lacZ) recombinant adenovirus or NS was injected into the sciatic nerve 0.5 cm distant from the sutured point respectively in different groups. The rats were transcardially perfused with 4% paraformaldehyde on postoperative 48 h, 7 d, 15 d and 30 d respectively and the spinal cords of L4 to L6 were harvested. X-gal staining, bcl-2 in situ hybridization and immunohistochemical staining, TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) staining and AChE (acetyl cholinesterase) histochemical staining were used. We observed that the exogenous lacZ gene was expressed in the spinal cord of Ad/lacZ group, and sense bcl-2 significantly decreased the number of apoptotic motoneurons and the decreasing degree of AChE activity of the motoneurons in the spinal cord induced by sciatic nerve axotomy and accelerate AChE activity recovery. However, antisense bcl-2 increased the number of apoptotic motoneurons and the decreasing degree of AChE activity of the motoneurons in the spinal cord induced by sciatic nerve axotomy and prolonged AChE activity recovery. These results demonstrate that exogenous bcl-2 may protect motor neurons from injury induced by peripheral nerve axotomy.

[Comparative study of the image observation of earlypostburn brain edema in severely burned dogs].

OBJECTIVE: To explore the diagnostic value of MRI and SPECT in early postburn brain edema in severely burned dogs. METHODS: Twenty-six mongrel dogs were randomized into control (n = 6) and burned groups in which every 5 dogs were allotted to each of following time points: 6, 12, 18 and 24 postburn hours (PBHs). The dogs in burn groups were inflicted with 50% TBSA of III degree skin burn and were infused with 5% glucose solution after 6 PBHs, so that severe early postburn brain edema was produced. MRI and SPECT were employed to observe dynamically the brain of dogs in all groups. The results were collected and compared with one another. RESULTS: The results indicated that with MRI brain morphological change of early brain edema could be shown as early as within 12 PBH and diffuse brain edema became more obvious with elapse of time. The changes might be difficult to be found by MRI when T(1)WISIR decreased below 10%. T(2)WI SIR increased by 8.29% at 24 PBH with blurred demarcation between the brain gray and white matters. There was diffused and progressive nuclide ((99)TCm-ECD) concentration in the brain tissue as shown by SPECT at 6 PBH. The radio-nuclide taking ratio increased significantly after 12 PBH, especially at 24 PBH (P < 0.01) when compared with that before burn. CONCLUSION: Combined application of MRI and SPECT could evidently increase sensitivity and specificity in the diagnosis of early postburn brain edema.

[Antigen expression and the biomechanical characteristics of the biologic blood vessel matrix].

To explore the changes of the antigen expression and the biomechanical characteristics of blood vessel in Banna little ear pig before and after trypsin treatment, and provide data for xenotransplantation and pig vessel using for tissue engineering. Geometric morphology and microstructure of pig cartoid artery were stuided quantitatively by histologic method and computer image analysis. The relationship between pressure and diameter was observed at different period of time before and after trypsin treatment. Affinity-immunohistochemistry assay was conducted to detect the expression of xenoantigens (alpha-Gal). The results showed that alpha-Gal antigen is only expressed in vascular endothelial cellsouly. There is no significant difference in blood vessel compliance. These demonstrate that the antigenicity of pig carotid artery is significantly reduced, however, the mechanical characteristics did not change significantly. We suppose that pig vessels treated by trypsin can be used as the substrate material for vascular tissue engineering.