Zhibo Han

Safety of mesenchymal stem cells for clinical application.

Mesenchymal stem cells (MSCs) hold great promise as therapeutic agents in regenerative medicine and autoimmune diseases, based on their differentiation abilities and immunosuppressive properties. However, the therapeutic applications raise a series of questions about the safety of culture-expanded MSCs for human use. This paper summarized recent findings about safety issues of MSCs, in particular their genetic stability in long-term in vitro expansion, their cryopreservation, banking, and the role of serum in the preparation of MSCs.

Proangiogenic Features of Mesenchymal Stem Cells and Their Therapeutic Applications

Mesenchymal stem cells (MSCs) have shown their therapeutic potency for treatment of cardiovascular diseases owing to their low immunogenicity, ease of isolation and expansion, and multipotency. As multipotent progenitors, MSCs have revealed their ability to differentiate into various cell types and could promote endogenous angiogenesis via microenvironmental modulation. Studies on cardiovascular diseases have demonstrated that transplanted MSCs could engraft at the injured sites and differentiate into cardiomyocytes and endothelial cells as well. Accordingly, several clinical trials using MSCs have been performed and revealed that MSCs may improve relevant clinical parameters in patients with vascular diseases. To fully comprehend the characteristics of MSCs, understanding their intrinsic property and associated modulations in tuning their behaviors as well as functions is indispensable for future clinical translation of MSC therapy. This review will focus on recent progresses on endothelial differentiation and potential clinical application of MSCs, with emphasis on therapeutic angiogenesis for treatment of cardiovascular diseases.

VCAM-1+ placenta chorionic villi-derived mesenchymal stem cells display potent pro-angiogenic activity.

IntroductionMesenchymal stem cells (MSCs) represent a heterogeneous cell population that is promising for regenerative medicine. The present study was designed to assess whether VCAM-1 can be used as a marker of MSC subpopulation with superior angiogenic potential.MethodsMSCs were isolated from placenta chorionic villi (CV). The VCAM-1+/− CV-MSCs population were separated by Flow Cytometry and subjected to a comparative analysis for their angiogenic properties including angiogenic genes expression, vasculo-angiogenic abilities on Matrigel in vitro and in vivo, angiogenic paracrine activities, cytokine array, and therapeutic angiogenesis in vascular ischemic diseases.ResultsAngiogenic genes, including HGF, ANG, IL8, IL6, VEGF-A, TGFβ, MMP2 and bFGF, were up-regulated in VCAM-1+CV-MSCs. Consistently, angiogenic cytokines especially HGF, IL8, angiogenin, angiopoitin-2, μPAR, CXCL1, IL-1β, IL-1α, CSF2, CSF3, MCP-3, CTACK, and OPG were found to be significantly increased in VCAM-1+ CV-MSCs. Moreover, VCAM-1+CV-MSCs showed remarkable vasculo-angiogenic abilities by angiogenesis analysis with Matrigel in vitro and in vivo and the conditioned medium of VCAM-1+ CV-MSCs exerted markedly pro-proliferative and pro-migratory effects on endothelial cells compared to VCAM-1−CV-MSCs. Finally, transplantation of VCAM-1+CV-MSCs into the ischemic hind limb of BALB/c nude mice resulted in a significantly functional improvement in comparison with VCAM-1−CV-MSCs transplantation.ConclusionsVCAM-1+CV-MSCs possessed a favorable angiogenic paracrine activity and displayed therapeutic efficacy on hindlimb ischemia. Our results suggested that VCAM-1+CV-MSCs may represent an important subpopulation of MSC for efficient therapeutic angiogenesis.

Transplantation of Human Placenta-Derived Mesenchymal Stem Cells Alleviates Critical Limb Ischemia in Diabetic Nude Rats

Neovasculogenesis induced by stem cell therapy is an innovative approach to improve critical limb ischemia (CLI) in diabetes. Mesenchymal stem cells (MSCs) are ideal candidates due to their angiogenic and immunomodulatory features. The aim of this study is to determine the therapeutic effects of human placenta-derived MSCs (P-MSCs) on diabetic CLI, with or without exogenous insulin administration, and the underlying mechanism of any effect. A series of in vitro experiments were performed to assess the stemness and vasculogenic activity of P-MSCs. P-MSCs were intramuscularly injected at two different doses with and without the administration of insulin. The efficacy of P-MSC transplantation was evaluated by ischemia damage score, ambulatory score, laser Doppler perfusion image (LDPI), capillary, and vascular density. In vivo imaging was applied to track the implanted P-MSCs. In vivo differentiation and in situ secretion of angiogenic cytokines were determined. In vitro experimental outcomes showed the differentiation potential and potent paracrine effect of P-MSCs. P-MSCs survived in vivo for at least 3 weeks and led to the acceleration of ischemia recovery, due to newly formed capillaries, increased arterioles, and secretion of various proangiogenic factors. P-MSCs participate in angiogenesis and vascularization directly through differentiation and cytokine expression.

Bone Marrow Is a Reservoir for Cardiac Resident Stem Cells.

Resident cardiac stem cells (CSCs) represent a responsive stem cell reservoir within the adult myocardium and have a significant function in myocardial homeostasis and injury. However, the distribution, origin, homing and possible therapeutic benefits of CSCs are still under discussion. Here we investigated whether bone marrow (BM) stem cells could contribute to repopulating the pool of CSCs in heart. The engraftment of BM cells in heart was detected at a low level after BM transplantation (BMT) and ischemia/reperfusion (I/R) could increase BM cells engraftment but not significant. We clarified that more than 50% CSCs are derived from BM and confirmed that BM-derived CSCs have similar characteristics with the host CSCs. Furthermore, we transplanted BM-derived CSCs into heart ischemia models and presented evidence for the first time that BM-derived CSCs can differentiate into cardiomyocytes in vivo. In conclusions, BM stem cells could be a potential back-up source of CSCs for restoring heart function after injury or maintaining homeostasis of CSCs.

Nitric oxide releasing hydrogel promotes endothelial differentiation of mouse embryonic stem cells

Transplantation of endothelial cells (ECs) holds great promise for treating various kinds of ischemic diseases. However, the major challenge in ECs-based therapy in clinical applications is to provide high quality and enough amounts of cells. In this study, we developed a simple and efficient system to direct endothelial differentiation of mouse embryonic stem cells (ESCs) using a controllable chitosan nitric oxide (NO)-releasing hydrogel (CS-NO). ESCs were plated onto the hydrogel culture system, and the expressions of differentiation markers were measured. We found that the expression of Flk-1 (early ECs marker) and VE-cadherin (mature ECs marker) increased obviously under the controlled NO releasing environment. Moreover, the Flk-1 upregulation was accompanied by the activation of the phospho-inositide-3 kinase (PI3K)/Akt signaling. We also found that in the presence of the PI3K inhibitor (LY294002), the endothelial commitment of ESCs was abolished, indicating the importance of Akt phosphorylation in the endothelial differentiation of ESCs. Interestingly, in the absence of NO, the activation of Akt phosphorylation alone by using AKT activator (SC-79) did not profoundly promote the endothelial differentiation of ESCs, suggesting an interdependent relationship between NO and the Akt phosphorylation in driving endothelial fate specification of ESCs. Taken together, we demonstrated that NO releasing in a continuous and controlled manner is a simple and efficient method for directing the endothelial differentiation of ESCs without adding growth factors. STATEMENT OF SIGNIFICANCE Fascinating data continues to show that artificial stem cell niche not only serve as a physical supporting scaffold for stem cells proliferation, but also as a novel platform for directing stem cell differentiation. Because of the lack of proper microenvironment for generating therapeutic endothelial cells (ECs) in vitro, the source of ECs for transplantation is the major limitation in ECs-based therapy to clinical applications. The current study established a feeder cell-free, 2-dimensional culture system for promoting the differentiation processes of embryonic stem cells (ESCs) committed to the endothelial lineage via using a nitric oxide (NO) controlled releasing hydrogel (CS-NO). Notably, the NO releasing from the hydrogel could selectively up-regulate Flk-1 (early ECs marker) and VE-cadherin (mature ECs marker) in the absence of growth factors, which was of crucial importance in the endothelial differentiation of ESCs. In summary, the current study proposes a simple and efficient method for directing the endothelial differentiation of ESCs without extra growth factors.

Comparison of Teratoma Formation between Embryonic Stem Cells and Parthenogenetic Embryonic Stem Cells by Molecular Imaging

With their properties of self-renewal and differentiation, embryonic stem (ES) cells hold great promises for regenerative therapy. However, teratoma formation and ethical concerns of ES cells may restrict their potential clinical applications. Currently, parthenogenetic embryonic stem (pES) cells have attracted the interest of researchers for its self-renewing and pluripotent differentiation while eliciting less ethic concerns. In this study, we established a model with ES and pES cells both stably transfected with a double-fusion reporter gene containing renilla luciferase (Rluc) and red fluorescent protein (RFP) to analyze the mechanisms of teratoma formation. Transgenic Vegfr2-luc mouse, which expresses firefly luciferase (Fluc) under the promoter of vascular endothelial growth factor receptor 2 (Vegfr2-luc), was used to trace the growth of new blood vessel recruited by transplanted cells. Bioluminescence imaging (BLI) of Rluc/Fluc provides an effective tool in estimating the growth and angiogenesis of teratoma in vivo. We found that the tumorigenesis and angiogenesis capacity of ES cells were higher than those of pES cells, in which VEGF/VEGFR2 signal pathway plays an important role. In conclusion, pES cells have the decreased potential of teratoma formation but meanwhile have similar differentiating capacity compared with ES cells. These data demonstrate that pES cells provide an alternative source for ES cells with the risk reduction of teratoma formation and without ethical controversy.

Intravenous injection of allogeneic umbilical cord-derived multipotent mesenchymal stromal cells reduces the infarct area and ameliorates cardiac function in a porcine model of acute myocardial infarction

BackgroundMultipotent mesenchymal stromal cell (MSC) therapy has been widely recognized as a feasible strategy for regenerating injured myocardial tissue. However, little is known about the efficacy of intravenous injection of allogeneic umbilical cord (UC) MSCs in preclinical models of porcine myocardial infarction.MethodsDifferent dosages of allogeneic UC-MSCs or the vehicle [phosphate-buffered saline (PBS)] were delivered intravenously into an acute myocardial infarction (AMI) porcine model twice after coronary ligation. Echocardiography was performed to examine the cardiac function and single photon emission computed tomography (SPECT) and positron emission tomography (PET)/computed tomography (CT) was performed to detect cardiac perfusion and nonviable myocardium. At the end of the experiment, 2,3,5-triphenyl-tetrazolium chloride (TTC) staining and Masson T staining were performed to determine the infarct area. The protein and gene expression levels associated with cardiac function, inflammation, and angiogenesis were examined by Western blot and real time polymerase chain reaction (PCR). In vivo trafficking of intravenous injection of allogeneic UC-MSCs enhanced green fluorescent protein (eGFP) was detected by real time PCR and immunofluorescence.ResultsAfter systemic delivery, allogeneic UC-MSCs were largely distributed in the lungs and some in the infracted myocardium. At week 8 following AMI, echocardiography demonstrated significantly improved fractional shortening in the high-dose (1.5 × 106 cells/kg) group. SPECT-PET/CT showed that UC-MSC treatment in both high and low doses markedly ameliorated the left ventricle (LV) infarct area but did not significantly improve the myocardial perfusion defect. LV remodeling was inhibited by UC-MSC therapy, as reflected by a marked reduction in rthe fibrosis area at basal, middle, and apical levels and reduced extracellular matrix deposition in the total myocardial area. Inflammatory biomarkers (tumor necrosis factor alpha and interleukin-6) were reduced and pro-angiogenesis factors (vascular endothelial growth factor and platelet/endothelial cell adhesion molecule 1) were augmented in the myocardial infarct and border area. High-dose UC-MSCs increased the connexin 43 (Cx43) (myocardium preservation) expression in remote area of the LV myocardium after AMI.ConclusionsIntravenous injection of UC-MSCs is a feasible and effective way to preserve LV function and ameliorate myocardial remodeling in porcine AMI. The cardioprotective effects of UC-MSCs were attributed to paracrine factors that appear to augment angiogenesis, limit inflammation, and preserve Cx43 gap junction.

Enhanced Therapeutic Effects of Mesenchymal Stem Cell-Derived Exosomes with an Injectable Hydrogel for Hindlimb Ischemia Treatment

Mesenchymal stem cell (MSC)-derived exosomes have been recognized as new candidates for cell-free treatment of various diseases. However, maintaining the retention and stability of exosomes over time in vivo after transplantation is a major challenge in the clinical application of MSC-derived exosomes. Here, we investigated if human placenta-derived MSC-derived exosomes incorporated with chitosan hydrogel could boost the retention and stability of exosomes and further enhance their therapeutic effects. Our results demonstrated that chitosan hydrogel notably increased the stability of proteins and microRNAs in exosomes, as well as augmented the retention of exosomes in vivo as confirmed by Gaussia luciferase imaging. In addition, we assessed endothelium-protective and proangiogenesis abilities of hydrogel-incorporated exosomes in vitro. Meanwhile, we evaluated the therapeutic function of hydrogel-incorporated exosomes in a murine model of hindlimb ischemia. Our data demonstrated that chitosan hydrogel could enhance the retention and stability of exosomes and further augment the therapeutic effects for hindlimb ischemia as revealed by firefly luciferase imaging of angiogenesis. The strategy used in this study may facilitate the development of easy and effective approaches for assessing and enhancing the therapeutic effects of stem cell-derived exosomes.

Interferon‑γ alters the microRNA profile of umbilical cord‑derived mesenchymal stem cells

Numerous studies have demonstrated that interferon-γ (IFN-γ) is an important inflammatory cytokine, which may activate the immunomodulatory abilities of mesenchymal stem cells (MSCs), and may influence certain other functions of these cells. MicroRNAs are small non-coding RNAs that regulate the majority of the biological functions of cells and are important in a variety of biological processes. However, few studies have been performed to investigate whether IFN-γ affects the microRNA profile of MSCs. The aim of the present study was to analyze the microRNA profile of MSCs derived from the umbilical cord (UC-MSCs) cultured in the presence or absence of IFN-γ (IFN-UC-MSCs). An array that detects 754 microRNAs was used to determine the expression profiles. Statistical analysis of the array data revealed that 8 microRNAs were significantly differentially expressed in UC-MSCs and IFN-UC-MSCs. Reverse transcription-quantitative polymerase chain reaction validated the differential expression of the 8 identified microRNAs. The target genes of the 8 microRNAs were predicted through two online databases, TargetScan and miRanda, and the predicted results were screened by bioinformatics analysis. The majority of the target genes were involved in the regulation of transcription, signal transduction, proliferation, differentiation and migration. These results may provide insight into the mechanism underlying the regulation of the biological functions of MSCs by IFN-γ, in particular the immunomodulatory activity.