Wei-Wen Lin

Enhancement of cell retention and functional benefits in myocardial infarction using human amniotic-fluid stem-cell bodies enriched with endogenous ECM

Stem cell transplantation may repair the infarcted heart. Despite the encouraging preliminary results, an optimal cell type used and low retention of the transplanted cells remain to be overcome. In this study, a multiwelled methylcellulose hydrogel system was used to cultivate human amniotic-fluid stem cells (hAFSCs) to form spherically symmetric cell bodies for cellular cardiomyoplasty. The grown hAFSC bodies enriched with extracellular matrices (ECM) were xenogenically transplanted in the peri-infarct area of an immune-suppressed rat, via direct intramyocardial injection. Results of bioluminescence imaging and real-time PCR revealed that hAFSC bodies could considerably enhance cell retention and engraftment in short-term and long-term observations, when compared with dissociated hAFSCs. Echocardiography and magnetic resonance imaging showed that the enhanced cell engraftment in the hAFSC-body group could significantly attenuate the progression of heart failure, improve the global function, and increase the regional wall motion. At the infarct, expressions of HGF, bFGF and VEGF were significantly up-regulated, an indication of the significantly increased vessel densities in the hearts treated with hAFSC bodies. The injected hAFSC bodies could undergo differentiation into angiogenic and cardiomyogenic lineages and contribute to functional benefits by direct regeneration. The aforementioned results demonstrate that hAFSC bodies can attenuate cell loss after intramuscular injection by providing an adequate physical size and offering an enriched ECM environment to retain the transplanted cells in the myocardium, thus improving heart function.

Cardiac repair with injectable cell sheet fragments of human amniotic fluid stem cells in an immune-suppressed rat model.

Direct intramyocardial injection of the desired cell types in a dissociated form is a common route of cell transplantation for repair of damaged myocardium. However, following injection of dissociated cells, a massive loss of transplanted cells has been reported. In this study, human amniotic fluid stem cells (hAFSCs) were used as the cell source for the fabrication of cell sheet fragments, using a thermo-responsive methylcellulose hydrogel system. The fabricated hAFSC sheet fragments preserved the endogenous extracellular matrices (ECM) and retained their cell phenotype. Test samples were xenogenically transplanted into the peri-ischemic area of an immune-suppressed rat model at 1 week after myocardial infarction (MI) induction. There were four treatment groups (n>=10): sham; saline; dissociated hAFSCs; and hAFSC sheet fragments. The results obtained in the echocardiography revealed that the group treated with hAFSC sheet fragments had a superior heart function to those treated with saline or dissociated hAFSCs. Due to their inherent ECM, hAFSC sheet fragments had a better ability of cell retention and proliferation than dissociated hAFSCs upon transplantation to the host myocardium. Additionally, transplantation of hAFSC sheet fragments stimulated a significant increase in vascular density, consequently contributing towards improved wall thickness and a reduction in the infarct size, when compared with dissociated hAFSCs. Our histological findings and qPCR analyses suggest that the transplanted hAFSCs can be differentiated into cardiomyocyte-like cells and cells of endothelial lineages and modulate expression of multiple angiogenic cytokines and cardiac protective factor with the potential to promote neo-vascularization, which evidently contributed to the improvement of ventricular function.

Spherically Symmetric Mesenchymal Stromal Cell Bodies Inherent with Endogenous Extracellular Matrices for Cellular Cardiomyoplasty

Cell transplantation via direct intramyocardial injection is a promising therapy for patients with myocardial infarction; however, retention of the transplanted cells at the injection sites remains a central issue following injection of dissociated cells. Using a thermoresponsive hydrogel system with a multiwell structure, we successfully developed an efficient technique to generate spherically symmetric bodies of mesenchymal stromal cells (MSCs) inherent with endogenous extracellular matrices (ECMs) for direct intramyocardial injection. After injection through a needle and upon transferring to another growth surface, the time required to attach, migrate, and proliferate was significantly shorter for the MSC bodies than the dissociated MSCs. Employing a syngeneic rat model with experimental myocardial infarction, an intramyocardial injection was conducted with a needle directly into the peri‐infarct areas. There were four treatment groups (n = 10): sham, phosphate‐buffered saline, dissociated MSCs, and MSC bodies. The results obtained in the echocardiography and catheterization measurements demonstrated that the MSC body group had a superior heart function to the dissociated MSC group. Histologically, it was found that MSC bodies could provide an adequate physical size to entrap into the interstices of muscular tissues and offer a favorable ECM environment to retain the transplanted cells intramuscularly. Additionally, transplantation of MSC bodies stimulated a significant increase in vascular density, thus improving the cardiac function. These results indicated that the spherically symmetric bodies of MSCs developed in the study may serve as a cell‐delivery vehicle and improve the efficacy of therapeutic cell transplantation. STEM CELLS 2009;27:724–732

Porous tissue grafts sandwiched with multilayered mesenchymal stromal cell sheets induce tissue regeneration for cardiac repair.

AIMS To provide the basis for uniform cardiac tissue regeneration, a spatially uniform distribution of adhered cells within a scaffold is a prerequisite. To achieve this goal, a bioengineered tissue graft consisting of a porous tissue scaffold sandwiched with multilayered sheets of mesenchymal stromal cells was developed. METHODS AND RESULTS This tissue graft (sandwiched patch) was used to replace the infarcted wall in a syngeneic Lewis rat model with an experimentally chronic myocardial infarction (MI). There were four treatment groups (n >/= 10): sham, MI, empty patch, and sandwiched patch. After a 7 day culture of the sandwiched patch, a tissue graft with relatively uniform cell concentrations was obtained. The cells were viable and tightly adhered to the tissue scaffold, as the endogenous extracellular matrix inherent with multilayered cell sheets can act as an adhesive agent for cell attachment and retention. At retrieval, the area of the empty patch was relatively enlarged, suggesting reduced structural support, while that of the sandwiched patch remained about the same (P = 0.56). In the immunofluorescent staining, host cells together with neo-microvessels were clearly observed in the empty patch; however, there were still a large number of unfilled pores within the patch. In the sandwiched patch, besides host cells, originally seeded cells were populated within the entire patch. No apparent evidence of apoptotic cell death was found in both studied patches. Thus, the sandwiched-patch-treated hearts demonstrated a better heart function to the empty-patch-treated hearts (P < 0.05). CONCLUSION The results demonstrated that this novel bioengineered tissue graft can serve as a useful cardiac patch to restore the dilated left ventricle and stabilize heart functions after MI.

Injectable PLGA porous beads cellularized by hAFSCs for cellular cardiomyoplasty

Cellular cardiomyoplasty has been limited by poor graft retention after cell transplantation. To ensure good retention of the engrafted cells, a microfluidic device was used to fabricate spherical porous beads of poly(D,L-lactic-co-glycolic acid) as a platform for cell delivery. The beads thus obtained had a relatively uniform size, a highly porous structure, and a favorably interconnected interior architecture, to facilitate the transportation of oxygen and nutrients. These porous beads were loaded with human amniotic fluid stem cells (hAFSCs) to generate cellularized microscaffolds. Live/dead assay demonstrated that most of the cells in the porous constructs were viable. The hAFSCs that were grown in beads formed a complex three-dimensional organization with well-preserved extracellular matrices (ECM) according to their porous structure. Retention of the administered beads was clearly identified at the site of engraftment following an experimentally induced myocardial infarction in a rat model. The results of echocardiography, magnetic resonance imaging, and histological analyses suggest that the transplantation of hAFSC beads into an infarcted heart could effectively maintain its gross morphology, prevent successive ventricular expansion, and thereby improve the post-infarcted cardiac function. Immunofluorescent staining revealed that the microenvironment that was provided by the infarcted myocardium might offer cues for the induction of the engrafted hAFSCs into angiogenic and cardiomyogenic lineages. Our results demonstrate that the cellularized beads with endogenously secreted ECM were of sufficient physical size to be entrapped in the interstitial tissues following transplantation, thereby benefiting the infarcted heart.

Vascularization and restoration of heart function in rat myocardial infarction using transplantation of human cbMSC/HUVEC core-shell bodies.

Cell transplantation is a promising strategy for therapeutic treatment of ischemic heart diseases. In this study, cord blood mesenchymal stem cells (cbMSCs) and human umbilical vein endothelial cells (HUVECs) in the form of core-shell bodies (cbMSC/HUVEC bodies) were prepared to promote vascularization and restore heart functions in an experimentally-created myocardial infarction (MI) rat model. Saline, cbMSC bodies and HUVEC bodies were used as controls. In vitro results indicated that cbMSC/HUVEC bodies possessed the capability of heterotypic assembly of cbMSCs and HUVECs into robust and durable tubular networks on Matrigel. The up-regulated gene expressions of VEGF and IGF-1 reflected the robust expansion of tubular networks; in addition, the augmented levels of SMA and SM22 suggested smooth muscle differentiation of cbMSCs, possibly helping to improve the durability of networks. Moreover, according to the in vivo echocardiographic, magnetic resonance and computed-tomographic results, transplantation of cbMSC/HUVEC bodies benefited post-MI dysfunction. Furthermore, the vascularization analyses demonstrated the robust vasculogenic potential of cbMSC/HUVEC bodies in vivo, thus contributing to the greater viable myocardium and the less scar region, and ultimately restoring the cardiac function. The concept of core-shell bodies composed of perivascular cells and endothelial cells may serve as an attractive cell delivery vehicle for vasculogenesis, thus improving the cardiac function significantly.

A translational approach in using cell sheet fragments of autologous bone marrow-derived mesenchymal stem cells for cellular cardiomyoplasty in a porcine model

Based on a porcine model with surgically created myocardial infarction (MI) as a pre-clinical scheme, this study investigates the clinical translation of cell sheet fragments of autologous mesenchymal stem cells (MSCs) for cellular cardiomyoplasty. MSC sheet fragments retaining endogenous extracellular matrices are fabricated using a thermo-responsive methylcellulose hydrogel system. Echocardiographic observations indicate that transplantation of MSC sheet fragments in infarcted hearts can markedly attenuate the adverse ventricular dilation and preserve the cardiac function post MI, which is in contrast to the controlled groups receiving saline or dissociated MSCs. Additionally, histological analyses suggest that administering MSC sheet fragments significantly prevents the scar expansion and left ventricle remodeling after MI. Immunohistochemistry results demonstrate that the engrafted MSCs can differentiate into endothelial cells and smooth muscle cells, implying that angiogenesis and the subsequent regional perfusion improvement is a promising mechanism for ameliorating post-infarcted cardiac function. However, according to the data recorded by an implantable loop recorder, the transplanted MSCs may provoke arrhythmia. Nevertheless, the proposed approach may potentially lead to the eventual translation of MSC-based therapy into practical and effective clinical treatments.

Intramuscular delivery of 3D aggregates of HUVECs and cbMSCs for cellular cardiomyoplasty in rats with myocardial infarction.

Cell-based therapeutic neovascularization is a promising method for treating ischemic disorders. In this work, human umbilical vein endothelial cells (HUVECs) were thoroughly premixed with cord-blood mesenchymal stem cells (cbMSCs) and cultivated to form three-dimensional (3D) cell aggregates for cellular cardiomyoplasty. In the in vitro study, tubular networks were formed at day 1 after the co-culturing of dissociated HUVECs and cbMSCs on Matrigel; however, as time progressed, the grown tubular networks regressed severely. Conversely, when 3D cell aggregates were grown on Matrigel, mature and stable tubular networks were observed over time, under the influence of their intensive cell-extracellular matrix (ECM) interactions and cell-cell contacts. 3D cell aggregates were transplanted into the peri-infarct zones of rats with myocardial infarction (MI) via direct intramyocardial injection. Based on our pinhole single photon emission computed tomography (SPECT) myocardial-perfusion observations, echocardiographic heart-function examinations and histological analyses, the engrafted 3D cell aggregates considerably enhanced the vascular densities and the blood flow recovery in the ischemic myocardium over those of their dissociated counterparts, thereby reducing the size of perfusion defects and restoring cardiac function. These results demonstrate that the intramuscular delivery of 3D cell aggregates of HUVECs/cbMSCs can be a valuable cell-based regenerative therapeutic strategy against MI.

Multimodality noninvasive imaging for assessing therapeutic effects of exogenously transplanted cell aggregates capable of angiogenesis on acute myocardial infarction.

Although the induction of neovascularization by cell-based approaches has demonstrated substantial potential in treating myocardial infarction (MI), the process of cell-mediated angiogenesis and its correlation with therapeutic mechanisms of cardiac repair remain elusive. In this work, three-dimensional (3D) aggregates of human umbilical vein endothelial cells (HUVECs) and cord-blood mesenchymal stem cells (cbMSCs) are constructed using a methylcellulose hydrogel system. By maximizing cell-cell and cell-ECM communications and establishing a hypoxic microenvironment in their inner cores, these cell aggregates are capable of forming widespread tubular networks together with the angiogenic marker αvβ3 integrin; they secret multiple pro-angiogenic, pro-survival, and mobilizing factors when grown on Matrigel. The aggregates of HUVECs/cbMSCs are exogenously engrafted into the peri-infarct zones of rats with MI via direct local injection. Multimodality noninvasive imaging techniques, including positron emission tomography, single photon emission computed tomography, and echocardiography, are employed to monitor serially the beneficial effects of cell therapy on angiogenesis, blood perfusion, and global/regional ventricular function, respectively. The myocardial perfusion is correlated with ventricular contractility, demonstrating that the recovery of blood perfusion helps to restore regional cardiac function, leading to the improvement in global ventricular performance. These experimental data reveal the efficacy of the exogenous transplantation of 3D cell aggregates after MI and elucidate the mechanism of cell-mediated therapeutic angiogenesis for cardiac repair.

Perforation of Cardiac Wall by Cement Embolism after Vertebral Augmentation for Thoracic Spinal Fracture: A Case Report

We describe a rare case of cardiac perforation caused by cement emboli after vertebral augmentation technique two months after the procedure. The patient was sent to a local hospital due to progressive dyspnea and chest tightness. Massive pericardial effusion was first diagnosed at the hospital, and therefore he was transferred to our hospitals emergency department. Multidetector computed tomography (MDCT) showed a string-like foreign body over the right atrium and ventricle which had apparently caused perforation of cardiac wall. Under the diagnosis of cement spike perforation, we performed percutaneous endovascular retrieval. We failed to extract the cement due to its fragile characteristic of the fragment and further migration of cement into the right middle lobe of lung was noted. The patients symptoms improved after the penetrating emboli was removed from the cardiac wall, and no symptoms of pulmonary embolism occurred. The patient was discharged 5 days later with aspirin treatment for prevention of pulmonary embolism.