Zhiyan Du

The influence of chitosan hydrogel on stem cell engraftment, survival and homing in the ischemic myocardial microenvironment

One challenge of cellular cardiomyoplasty for myocardial infarction (MI) is how to improve MI microenvironment to facilitate stem cell engraftment, survival and homing for myocardial repair. The application of injectable hydrogels is an effective strategy. However, it has not been thoroughly investigated on the role of the injectable scaffolds, in improving MI microenvironment, providing space and guidance for cell survival, engraftment and homing. We explored an injectable chitosan hydrogel for stem cell delivery into ischemic heart and investigated the beneficial effects and mechanisms of the hydrogel. In vitro, H(2)O(2)-treatment was used to mimic reactive oxygen species (ROS) microenvironment. The influence of ROS and protection of chitosan components on adipose-derived mesenchymal stem cells (ADSCs) was analyzed too. In vivo, MI was induced by the left anterior descending artery ligation in SD rats. PBS, chitosan hydrogel, ADSC/PBS and ADSC/chitosan hydrogel were injected into the border of infracted hearts respectively. Multi-techniques were used to assess the beneficial effects of chitosan hydrogel after transplantation. We observed that ROS generated by ischemia would impair ADSC adhesion molecules, including integrin-related adhesion molecules integrin αV and β1, focal adhesion-related molecules p-FAK and p-Src, and corresponding ligands of host myocardium ICAM1 and VCAM1. Chitosan hydrogel could rescue these molecules through ROS scavenging and recruit key chemokine for stem cell homing, such as SDF-1. The results suggest that chitosan hydrogel could improve MI microenvironment, enhance stem cell engraftment, survival and homing in ischemic heart through ROS scavenging and chemokine recruitment, contributing to myocardial repair.

The tumourigenicity of iPS cells and their differentiated derivates.

Induced pluripotent stem cell (iPSC) provides a promising seeding cell for regenerative medicine. However, iPSC has the potential to form teratomas after transplantation. Therefore, it is necessary to evaluate the tumorigenic risks of iPSC and all its differentiated derivates prior to use in a clinical setting. Here, murine iPSCs were transduced with dual reporter gene consisting of monomeric red fluorescent protein (mRFP) and firefly luciferase (Fluc). Undifferentiated iPSCs, iPSC derivates from induced differentiation (iPSC‐derivates), iPSC‐derivated cardiomyocyte (iPSC‐CMs) were subcutaneously injected into the back of nude mice. Non‐invasive bioluminescence imaging (BLI) was longitudinally performed at day 1, 7, 14 and 28 after transplantation to track the survival and proliferation of transplanted cells. At day 28, mice were killed and grafts were explanted to detect teratoma formation. The results demonstrated that transplanted iPSCs, iPSC‐derivates and iPSC‐CMs survived in receipts. Both iPSCs and iPSC‐derivates proliferated dramatically after transplantation, while only slight increase in BLI signals was observed in iPSC‐CM transplanted mice. At day 28, teratomas were detected in both iPSCs and iPSC‐derivates transplanted mice, but not in iPSC‐CM transplanted ones. In vitro study showed the long‐term existence of pluripotent cells during iPSC differentiation. Furthermore, when these cells were passaged in feeder layers as undifferentiated iPSCs, they would recover iPSC‐like colonies, indicating the cause for differentiated iPSCs tumourigenicity. Our study indicates that exclusion of tumorigenic cells by screening in addition to lineage‐specific differentiation is necessary prior to therapeutic use of iPSCs.

The use of chitosan based hydrogel for enhancing the therapeutic benefits of adipose-derived MSCs for acute kidney injury

Transplantation of mesenchymal stem cells (MSCs) has been reported a great therapeutic potential for acute kidney injury (AKI). However, the therapeutic benefits are limited due to the low retention and survival of transplanted cells within target sites. In this study, thermosensitive chitosan chloride (CSCl) hydrogel was explored as injectable scaffold for adipose-derived MSCs (ADMSCs) delivery into ischemia/reperfusion (I/R) induced acute kidney injury (AKI). Thermosensitive CSCl hydrogels with/without ADMSCs were injected into the I/R site of rat AKI models. Dihydroethidium staining was used to detect the number of ROS in vivo. In order to track ADMSCs in vivo, ADMSCs were transfected with firefly luciferase and monomeric red fluorescent protein reporter genes (fluc-mrfp). The retention and survival of ADMSC were assessed using bioluminescence imaging, differentiation behaviors of ADMSCs were investigated using immunofluorescent and immunohistochemical staining. Proliferation and apoptosis of host renal cell in vivo were characterized by PCNA and TUNEL staining. Results suggested that CSCl hydrogels could improve the retention and survival of grafted ADMSCs, moreover, CSCl hydrogels could enhance the proliferation activity and reduce apoptosis of host renal cells. At 4 weeks, significant improvement of the renal function, microvessel density and tubular cell proliferation were observed in CSCl hydrogels with ADMSCs groups. Therefore, the application of thermosensitive CSCl hydrogel as scaffold for ADMSCs delivery into renal region could resolve the main obstacle of cell transplantation for acute kidney injury (AKI). Therefore, CSCl hydrogel is a potential cell carrier for treatment of AKI.

Immunohistochemical characterization and functional identification of mammary gland telocytes in the self-assembly of reconstituted breast cancer tissue in vitro

Telocyte (TC) as a special stromal cell exists in mammary gland and might play an important role in the balance of epithelium‐stroma of mammary gland. Considering that different types of breast interstitial cells influence the development and progression of breast cancer, TCs may have its distinct role in this process. We here studied the roles of TCs in the self‐assembly of reconstituted breast cancer tissue. We co‐cultured primary isolated TCs and other breast stromal cells with breast cancer EMT‐6 cells in collagen/Matrigel scaffolds to reconstitute breast cancer tissue in vitro. Using histology methods, we investigated the immunohistochemical characteristics and potential functions of TCs in reconstituted breast cancer tissue. TCs in primary mammary gland stromal cells with long and thin overlapping cytoplasmic processes, expressed c‐kit/CD117, CD34 and vimentin in reconstitute breast cancer tissue. The transmission electron microscopy showed that the telocyte‐like cells closely communicated with breast cancer cells as well as other stromal cells, and might serve as a bridge that directly linked the adjacent cells through membrane‐to‐membrane contact. Compared with cancer tissue sheets of EMT‐6 alone, PCNA proliferation index analysis and TUNEL assay showed that TCs and other breast stromal cells facilitated the formation of typical nest structure, promoted the proliferation of breast cancer cells, and inhibited their apoptosis. In conclusion, we successfully reconstituted breast cancer tissue in vitro, and it seems to be attractive that TCs had potential functions in self‐assembly of EMT‐6/stromal cells reconstituted breast cancer tissue.

Melatonin protects ADSCs from ROS and enhances their therapeutic potency in a rat model of myocardial infarction.

Myocardial infarction (MI) is a major cause of death and disability worldwide. In the last decade, mesenchymal stem cells (MSCs) based cell therapy has emerged as a promising therapeutic strategy. Although great advance have been made using MSCs to treat MI, the low viability of transplanted MSCs severely limits the efficiency of MSCs therapy. Here, we show evidence that ex vivo pre‐treatment with melatonin, an endogenous hormone with newly found anti‐oxidative activity, could improve survival and function of adipose tissue derived MSCs (ADSCs) in vitro as well as in vivo. ADSCs with 5 μM melatonin pre‐treatment for 24 hrs showed increased expression of the antioxidant enzyme catalase and Cu/Zn superoxide dismutase (SOD‐1), as well as pro‐angiogenic and mitogenic factors like insulin‐like growth factor 1, basic fibroblast growth factor, hepatocyte growth factor (HGF), epidermal growth factor. Furthermore, melatonin pre‐treatment protected MSCs from reactive oxygen species (ROS) induced apoptosis both directly by promoting anti‐apoptosis kinases like p‐Akt as well as blocking caspase cascade, and indirectly by restoring the ROS impaired cell adhesion. Using a rat model of MI, we found that melatonin pre‐treatment enhanced the viability of engrafted ADSCs, and promoted their therapeutic potency. Hopefully, our results may shed light on the design of more effective therapeutic strategies treating MI by MSCs in clinic.

Self-assembly of renal cells into engineered renal tissues in collagen/Matrigel scaffold in vitro.

To tissue engineer a kidney is a formidable task because of the complex cell composition and structures in the kidney. This study reconstructed renal tissues using mixed renal cells in collagen/Matrigel® scaffolds in vitro. Neonatal rat renal cells were seeded in collagen I supplemented with Matrigel in a casting mold that could exert static stretch when the renal constructs contracted. During in vitro culture, the renal constructs were observed under microscope and analyzed by histological and immunofluorescent examinations. Results showed that the mixed renal cells reconstituted renal tubular and glomeruli‐like structures with different appearances at varying developmental stages. Tubular structures were formed by CK18‐positive cells with similar appearances lining the surrounding hollow centres. The glomeruli‐like structures were tufts of cell aggregates containing Flk‐1‐positive cells. These results show that neonatal rat renal cells self‐assembled into engineered renal tissues containing both tubules and glomeruli‐like structures when cultured in 3D collagen/Matrigel scaffold in vitro. Copyright

Molecular Imaging of Induced Pluripotent Stem Cell Immunogenicity with In Vivo Development in Ischemic Myocardium

Whether differentiation of induced pluripotent stem cells (iPSCs) in ischemic myocardium enhances their immunogenicity, thereby increasing their chance for rejection, is unclear. Here, we dynamically demonstrated the immunogenicity and rejection of iPSCs in ischemic myocardium using bioluminescent imaging (BLI). Murine iPSCs were transduced with a tri-fusion (TF) reporter gene consisting of firefly luciferase-red fluorescent protein-truncated thymidine kinase (fluc-mrfp-tTK). Ascorbic acid (Vc) were used to induce iPSCs to differentiate into cardiomyocytes (CM). iPSCs and iPS-CMs were intramyocardially injected into immunocompetent or immunosuppressed allogenic murine with myocardial infarction. BLI was performed to track transplanted cells. Immune cell infiltration was evaluated by immunohistochemistry. Syngeneic iPSCs were also injected and evaluated. The results demonstrated that undifferentiated iPSCs survived and proliferated in allogenic immunocompetent recipients early post-transplantation, accompanying with mild immune cell infiltration. With in vivo differentiation, a progressive immune cell infiltration could be detected. While transplantation of allogenic iPSC-CMs were observed an acute rejection from receipts. In immune-suppressed recipients, the proliferation of iPSCs could be maintained and intramyocardial teratomas were formed. Transplantation of syngeneic iPSCs and iPSC-CMs were also observed progressive immune cell infiltration. This study demonstrated that iPSC immunogenicity increases with in vivo differentiation, which will increase their chance for rejection in iPSC-based therapy.

The stem cell adjuvant with Exendin-4 repairs the heart after myocardial infarction via STAT3 activation

The poor survival of cells in ischaemic myocardium is a major obstacle for stem cell therapy. Exendin‐4 holds the potential of cardioprotective effect based on its pleiotropic activity. This study investigated whether Exendin‐4 in conjunction with adipose‐derived stem cells (ADSCs) could improve the stem cell survival and contribute to myocardial repairs after infarction. Myocardial infarction (MI) was induced by the left anterior descending artery ligation in adult male Sprague‐Dawley rats. ADSCs carrying double‐fusion reporter gene [firefly luciferase and monomeric red fluorescent protein (fluc‐mRFP)] were quickly injected into border zone of MI in rats treated with or without Exendin‐4. Exendin‐4 enhanced the survival of transplanted ADSCs, as demonstrated by the longitudinal in vivo bioluminescence imaging. Moreover, ADSCs adjuvant with Exendin‐4 decreased oxidative stress, apoptosis and fibrosis. They also improved myocardial viability and cardiac function and increased the differentiation rates of ADSCs into cardiomyocytes and vascular smooth muscle cells in vivo. Then, ADSCs were exposed to hydrogen peroxide/serum deprivation (H2O2/SD) to mimic the ischaemic environment in vitro. Results showed that Exendin‐4 decreased the apoptosis and enhanced the paracrine effect of ADSCs. In addition, Exendin‐4 activated signal transducers and activators of transcription 3 (STAT3) through the phosphorylation of Akt and ERK1/2. Furthermore, Exendin‐4 increased the anti‐apoptotic protein Bcl‐2, but decreased the pro‐apoptotic protein Bax of ADSCs. In conclusion, Exendin‐4 could improve the survival and therapeutic efficacy of transplanted ADSCs through STAT3 activation via the phosphorylation of Akt and ERK1/2. This study suggests the potential application of Exendin‐4 for stem cell–based heart regeneration.

Transplantation of co-microencapsulated hepatocytes and HUVECs for treatment of fulminant hepatic failure

Purpose: Microencapsulated hepatocytes might solve immunological rejection, broadening a new perspective for the treatment of fulminant hepatic failure (FHF). However, the transplantation of microcapsulated hepatocytes is limited by low cell viability Nevertheless, the co-microencapsulation of hepatocytes and human umbilical vein endothelial cells (HUVECs) may make the treatment of FHF more promising. Methods: We prepared the microcapsules using the high-voltage electrostatic droplet spray method, transplanted the empty microcapsules, isolated hepatocytes, microcapsulated hepatocytes, and co-microencapsulated hepatocytes and HUVEC intraperitoneally into rat models of FHF induced by D-aminogalactose (D-gal). After 1, 3, and 7 days, and 2, 3, and 4 weeks posttransplantation, we calculated the mortality and assessed alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (ALB) levels in the serum of the model; evaluated the integrality and recovery of microcapsules; and stained with hematoxylin and eosin (H&E) the recovered microcapsules as well as the liver of the FHF rats. Results: Hepatocyte-specific functions, including the levels of ALT, AST, and ALB in the serum of the co-microencapsulation group, were significantly better than those in the other groups (p<0.05) from 2 to 4 weeks after transplantation. Moreover, cotransplantation of the microencapsulated hepatocytes and HUVECs decreased the mortality rate of the FHF rats. The recovered microcapsules were intact, and recovery was up to 90%. H&E staining showed that the microencapsulated cells were still alive, and the liver tissues had started to recover after 4 weeks posttransplantation. Conclusion: The microcapsules have good biocompatibility and immunoprotection to protect the hepatocytes from immunological rejection. Cotransplantation of the microencapsulated hepatocytes and HUVECs could decrease mortality rates and improve liver function in FHF.

Evaluating cell migration in vitro by the method based on cell patterning within microfluidic channels

Cell migration is an early‐stage and critical step for cancer metastasis. The most common approach to monitor this process is wound‐healing assay. However, this traditional method has some unavoidable limitations. We observed that simply scratching the monolayer of cultured cells might cause local cell damage around the injury line. The cells along the scratched border seemed to be irritated and exhibited abnormal distribution of cytoskeleton reassembly with protruding “cell islands” and “pseudopodia” during wound healing, which might potentially affect the assessment of cell migration behavior. Herein, we applied a microfluidic device that mechanically constrained cells seeded in a designed pattern inside microchannels, and monitored cell movement in a way of mimicking the natural microenvironment of cancerous tissues. We illustrated the capacity of this simple method to probe cellular migration behaviors and to screen some biological active agents that reflected in their influence on cellular motility.