Yibing Guo

Decellularization and Recellularization of Rat Livers With Hepatocytes and Endothelial Progenitor Cells.

Whole-organ decellularization has been identified as a promising choice for tissue engineering. The aim of the present study was to engineer intact whole rat liver scaffolds and repopulate them with hepatocytes and endothelial progenitor cells (EPCs) in a bioreactor. Decellularized liver scaffolds were obtained by perfusing Triton X-100 with ammonium hydroxide. The architecture and composition of the original extracellular matrix were preserved, as confirmed by morphologic, histological, and immunolabeling methods. To determine biocompatibility, the scaffold was embedded in the subcutaneous adipose layer of the back of a heterologous animal to observe the infiltration of inflammatory cells. Hepatocytes were reseeded using a parenchymal injection method and cultured by continuous perfusion. EPCs were reseeded using a portal vein infusion method. Morphologic and functional examination showed that the hepatocytes and EPCs grew well in the scaffold. The present study describes an effective method of decellularization and recellularization of rat livers, providing the foundation for liver engineering and the development of bioartificial livers.

Synthesis and protective effects of aralkyl alcoholic 2-acetamido-2-deoxy-β-D-pyranosides on hypoglycemia and serum limitation induced apoptosis in PC12 cell.

Neuroprotective agents have been in the focus of attention in the treatment of ischemic stroke. Salidroside, a phenylpropanoid glycoside isolated from Rhodiola rosea L., possessed a wide range of biological activities, especially neuroprotection. In an attempt to improve neuroprotective effects of new salidroside analogs for ischemic stroke, a series of novel aralkyl alcoholic 2-acetamido-2-deoxy-β-d-pyranosides were synthesized and their protective activities against the hypoglycemia and serum limitation induced cell death in rat pheochromocytoma cells (PC12 cells) were studied. Most compounds showed strong neuroprotective effects, especially for 4g and 4h, which exhibited a great potency superior to salidroside. MTT assay, Hoechst 33342 staining, and flow cytometry with annexin V/PI staining collectively showed that pretreatment with 4g and 4h attenuated cell viability loss and apoptotic cell death in cultured PC12 cells. Caspase-3 colorimetric assay and Rhodamine 123 staining revealed the changes in expression levels of caspase-3 and mitochondrial membrane potential in PC12 cells on exposure to hypoglycemia and serum limitation with and without 4g and 4h pretreatment, respectively. All the results suggested that 4g and 4h protects the PC12 cells against hypoglycemia and serum limitation induced apoptosis possibly by modulation of apoptosis-related gene expression and restoration of the mitochondrial membrane potential. Therefore, these novel findings may provided a new framework for the design of new aralkyl alcoholic 2-acetamido-2-deoxy-β-d-pyranosides as neuroprotective agents for treating cerebral ischemic stroke and neurodegenerative diseases.

Biocompatibility evaluation of electrospun silk fibroin nanofibrous mats with primarily cultured rat hippocampal neurons.

In this study, electrospinning was performed to fabricate silk fibroin (SF) nanofibrous mats, which were used as substrates for in vitro culture of rat hippocampal neurons. The light and electron micrographs demonstrated that the electrospun SF nanofibrous mat supported the survival and growth of the attached hippocampal neurons. MTT assay and immunocytochemistry in couple with Western blot analysis respectively indicated there was no significant difference in both the cell viability and expression levels of some proteins, including GAP-43, MAP-2, NF, and β-tubulin, between hippocampal neurons cultured in the electrospun SF nanofibrous mat extract and in plain neuronal medium. Our results indicated that electrospun SF nanofibrous mats were biocompatible to primary culture of hippocampal neurons without cytotoxic effects on the cell phenotype and functions, raising a potential possibility of using these mats for CNS therapeutic applications.

3D Culture of MIN-6 Cells on Decellularized Pancreatic Scaffold: In Vitro and In Vivo Study

Type 1 diabetes is an autoimmune disease which is due to the lack of β cells. The ideal therapy to cure the disease is pancreas transplantation, but its application is confined to a limited number of people due to the shortage of organ and the need for life-long immunosuppression. Regenerative medicine methods such as a tissue engineered pancreas seem to provide a useful method. In order to construct a microenvironment similar to the native pancreas that is suitable for not only cell growth but also cellular function exertion, a decellularized mouse pancreas was used as a natural 3D scaffold in this experiment. MIN-6 β cells were planted in the bioscaffold. The cell engraftment was verified by HE staining and SEM. Immunostaining procedures were performed to confirm the normal function of the engrafted cells. qRT-PCR demonstrated that insulin gene expression of the recellularized pancreas was upregulated compared with conventional plate-cultured cells. In vivo experiment was also accomplished to further evaluate the function of the recellularized bioscaffold and the result was inspiring. And beyond doubt this will bring new hope for type 1 diabetic patients.

Three-dimensional culture of mouse pancreatic islet on a liver-derived perfusion-decellularized bioscaffold for potential clinical application.

The cutting-edge technology of three-dimensional liver decellularized bioscaffold has a potential to provide a microenvironment that is suitable for the resident cells and even develop a new functional organ. Liver decellularized bioscaffold preserved the native extracellular matrix and three-dimensional architecture in support of the cell culture. The goal of this study was to discover if three-dimensional extracellular matrix derived from mouse liver could facilitate the growth and maintenance of physiological functions of mouse isolated islets. We generated a whole organ liver decellularized bioscaffold which could successfully preserve extracellular matrix proteins and the native vascular channels using 1% Triton X-100/0.1% ammonium protocol. To evaluate the potential of decellularized liver as a scaffold for islets transplantation, the liver decellularized bioscaffold was infused with mouse primary pancreatic islets which were obtained through Collagenase P digestion protocol. Its yield, morphology, and quality were estimated by microscopic analysis, dithizone staining, insulin immunofluorescence and glucose stimulation experiments. Comparing the three-dimensional culture in liver decellularized bioscaffold with the orthodoxy two-dimensional plate culture, hematoxylin-eosin staining, immunohistochemistry, and insulin gene expression were tested. Our results demonstrated that the liver decellularized bioscaffold could support cellular culture and maintenance of cell functions. In contrast with the conventional two-dimensional culture, three-dimensional culture system could give rise to an up-regulated insulin gene expression. These findings demonstrated that the liver bioscaffold by a perfusion-decellularized technique could serve as a platform to support the survival and function of the pancreatic islets in vitro. Meanwhile three-dimensional culture system had a superior role in contrast with the two-dimensional culture. This study advanced the field of regenerative medicine towards the development of a liver decellularized bioscaffold capable of forming a neo-organ and could be used as potential clinical application.

Culture of iPSCs Derived Pancreatic β-Like Cells In Vitro Using Decellularized Pancreatic Scaffolds: A Preliminary Trial

Diabetes mellitus is a disease which has affected 415 million patients in 2015. In an effort to replace the significant demands on transplantation and morbidity associated with transplantation, the production of β-like cells differentiated from induced pluripotent stem cells (iPSCs) was evaluated. This approach is associated with promising decellularized scaffolds with natural extracellular matrix (ECM) and ideal cubic environment that will promote cell growth in vivo. Our efforts focused on combining decellularized rat pancreatic scaffolds with mouse GFP+-iPSCs-derived pancreatic β-like cells, to evaluate whether decellularized scaffolds could facilitate the growth and function of β-like cells. β-like cells were differentiated from GFP+-iPSCs and evaluated via cultivating in the dynamic circulation perfusion device. Our results demonstrated that decellularized pancreatic scaffolds display favorable biochemical properties. Furthermore, not only could the scaffolds support the survival of β-like cells, but they also accelerated the expression of the insulin as compared to plate-based cell culture. In conclusion, these results suggest that decellularized pancreatic scaffolds could provide a suitable platform for cellular activities of β-like cells including survival and insulin secretion. This study provides preliminary support for regenerating insulin-secreting organs from the decellularized scaffolds combined with iPSCs derived β-like cells as a potential clinical application.

Rab8a/Rab11a regulate intercellular communications between neural cells via tunneling nanotubes.

Tunneling nanotubes (TNTs) are F-actin-based membrane tubes, and can form between cultured cells and within vital tissues. TNTs mediate intercellular communications that range from electrical signaling to the transfer of organelles. Following peripheral nerve injury, the orchestrated intercellular communications among neural and non-neural cells are required for effective nerve regeneration. It remains unknown whether TNTs exist between neural cells in the peripheral nerve system and how TNTs affect neural regeneration. To address these interesting questions, we investigated the transfer of neurotropic factors, membrane protein, cytoplasmic protein, mitochondria and RNA in functional TNTs formed between cultured Schwann cells (SCs). TNT-like structures were increased not only in cultured SCs after exposure to serum depletion but also in longitudinal sections of proximal sciatic nerve stump harvested after rat peripheral nerve transection. Meanwhile, downregulation of Rab8a or Rab11a in cultured SCs inhibited the formation of functional TNTs and vesicle transfer and led to decrease in cell migration, increase in SCs apoptosis. Likewise, knockdown of Rab8a or Rab11a in primary SCs also suppressed axonal outgrowth from co-cultured dorsal root ganglion (DRG) neurons. Overall, our results suggested that the gene of Rab8a or Rab11a might be involved in the formation of TNTs structures in the peripheral nerve system, while TNTs structures were likely to affect peripheral nerve regeneration through the regulation of neural cell communications.

Vascularization of pancreatic decellularized scaffold with endothelial progenitor cells

Vascularization remains a large obstacle for creating a functional pancreas-tissue equivalent for transplantation. In this study, a pre-vascularized pancreatic decellularized scaffold was prepared through endothelializing with endothelial progenitor cells (EPCs) in a bioreactor, and the ability to regenerate new blood vessels was detected in vivo. Initially, pancreases of Sprague–Dawley (SD) rats were perfused with 1% Triton X-100 and 0.1% ammonium hydroxide to remove the cellular components while the intact vascular network was preserved. Then, the decellularized scaffold was reseed with EPCs, which were primarily characterized by dual staining for dil-labeled acetylated low-density lipoprotein (Dil-acLDL) and fluorescein isothiocyanate labeled ulex europaeus agglutinin 1 (FITC-UEA-1), to reconstruct the vascular network. Thus, a scaffold covered with EPCs in the vessel structure was created. After that, the scaffold was transplanted into the rat in vivo to observe the anastomosis with the host vascular network. The results showed that EPCs can be located around the blood vessel wall, and re-endothelialized scaffold connected with the host through new blood vessel formation earlier than the control group (p < 0.05). These findings all indicated that the pancreatic decellularized scaffold endothelialized with EPCs may be further applied to solve the problem of blood supply and support the function of insulin-secreting cells after in vivo transplantation.

Transcriptome analysis of the induced pluripotent stem cell (iPSC)-derived pancreatic β-like cell differentiation.

Diabetes affects millions of people worldwide, and β-cell replacement is one of the promising new strategies for treatment. Induced pluripotent stem cells (iPSCs) can differentiate into any cell type, including pancreatic β cells, providing a potential treatment for diabetes. However, the molecular mechanisms underlying the differentiation of iPSC-derived β cells have not yet been fully elucidated. Here, we generated pancreatic β-like cells from mouse iPSCs using a 3-step protocol and performed deep RNA sequencing to get a transcriptional landscape of iPSC-derived pancreatic β-like cells during the selective differentiation period. We then focused on the differentially expressed genes (DEGs) during the time course of the differentiation period, and these genes underwent Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis. In addition, gene-act networks were constructed for these DEGs, and the expression of pivotal genes detected by quantitative real-time polymerase chain reaction was well correlated with RNA sequence (RNA-seq). Overall, our study provides valuable information regarding the transcriptome changes in β cells derived from iPSCs during differentiation, elucidates the biological process and pathways underlying β-cell differentiation, and promotes the identification and functional analysis of potential genes that could be used for improving functional β-cell generation from iPSCs.

HSPA12B regulates SSeCKS-mediated astrocyte inflammatory activation in neuroinflammation.

Reactive astrocytosis has been considered either beneficial or detrimental effection in neuroinflammatory disease. HSPA12B, a new member belongs to the 70-kDa family of heat shock proteins (HSP70) which could modulate inflammatory response, also shows an connection with the astrocyte activation. Recently, it was reported that Src-Suppressed-C Kinase Substrate (SSeCKS) was detected in heat shock protein A12B (HSPA12B) interacting proteins using a yeast 2-hybrid system. SSeCKS, a major Lipopolysaccharide (LPS) response protein, has been involved in regulating astrocyte activation via production of proinflammatory factor in CNS inflammation. In this study, we found HSPA12B might regulate the expression and activity of SSeCKS to promote astrocyte inflammatory activation and release of inflammatory mediators, such as TNF-α and IL-1β in spinal cord primary astroglial cultures exposed to LPS treatment. The promoting mechanism of interaction between HSPA12B and SSeCKS on LPS-induced astrocyte activation was mediated via the activation of JNK and p38 signaling pathways but not ERK1/2 MAPK signaling pathway. HSPA12B binded to SSeCKS via its both N terminus consisted of amino acids 1-330 and C terminus consisted of amino acids 1278-1596. And, in vivo, we confirmed the interaction between HSPA12B and SSeCKS of astrocyte activation in the pathogenesis of EAE. The regulatory mechanisms of HSPA12B-SSeCKS interaction may possibly be the key therapeutic strategy of neuroinflammatory disease.