Development of composite functional tissue sheets using hiPSC-CMs and hADSCs to improve the cardiac function after myocardial infarction.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been widely used in therapy of ischemic heart disease. However, there are still remaining issues that limit the therapeutic efficacy, such as immune rejection and low retention of hiPSC-CMs. Human adipose mesenchymal stromal cells (hADSCs) have been reported to be able to regulate the immune response, promote angiogenesis and promote the maturation of hiPSC-CMs. In this study, we co-cultured these two types of cells on fiber scaffold made of biodegradable poly (D,L-lactic-co-glycolic acid) (PLGA) polymer for several days to develop a composited 3D cardiac tissue sheet. As expected, the cells formed 231.00 ± 15.14 µm thickness tissue, with improved organization, alignment, ECM condition, contractile ability, and paracrine function compared to culture hiPSC-CMs only on PLGA fiber. Furthermore, the composited 3D cardiac tissue sheet significantly promoted the engraftment and survival after transplantation. The composited 3D cardiac tissue sheet also increased cardiac function, attenuated ventricular remodeling, decreased fibrosis, and enhanced angiogenesis in rat myocardial infarction model, indicating that this strategy wound be a promising therapeutic option in the clinical scenario.

CD8+ T cell-mediated rejection of allogenic human-induced pluripotent stem cell-derived cardiomyocyte sheets in human PBMC-transferred NOG MHC double knockout mice.

BACKGROUND: Transplantation of human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) has emerged as a promising therapy to treat end-stage heart failure. However, the immunogenicity of hiPS-CMs in transplanted patients has not been fully elucidated. Thus, in vivo models are required to estimate immune responses against hiPS-CMs in transplant recipients. METHODS: We transferred human peripheral blood mononuclear cells (hPBMCs) into NOD/Shi-scid IL-2rgnull (NOG) MHC class I/II double knockout (NOG-ΔMHC) mice, which were reported to accept hPBMCs without xenogeneic-graft-versus-host disease (xeno-GVHD). Then, hiPS-CM sheets generated from the hiPS cell line 201B7 harboring a luciferase transgene were transplanted into the subcutaneous space of NOG-ΔMHC mice. Graft survival was monitored by bioluminescent images using a Xenogen In Vivo Imaging System. RESULTS: The human immune cells were engrafted for more than 3 months in NOG-ΔMHC mice without lethal xeno-GVHD. The hiPS-CMs expressed a moderate level of human leukocyte antigen (HLA)-class I, but not HLA-class II, molecules even after interferon-gamma (IFN-γ) stimulation. Consistently, the allogenic IFN-γ-treated hiPS-CMs induced weak CD8+ but not CD4+ T cell responses in vitro. hiPS-CM sheets disappeared approximately 17 to 24 days after transplantation in hPBMC-transferred NOG-ΔMHC mice, and CD8+ T cell depletion significantly prolonged graft survival, similar to what was observed following tacrolimus treatment. CONCLUSIONS: hiPS-CMs are less immunogenic in vitro but induce sufficient CD8+ T cell-mediated immune responses for graft rejection in vivo.

Human induced pluripotent stem cell-derived closed-loop cardiac tissue for drug assessment.

Human iPSC-derived cardiomyocytes (hiPSC-CMs) exhibit functional immaturity, potentially impacting their suitability for assessing drug proarrhythmic potential. We previously devised a traveling wave (TW) system to promote maturation in 3D cardiac tissue. To align with current drug assessment paradigms (CiPA and JiCSA), necessitating a 2D monolayer cardiac tissue, we integrated the TW system with a multi-electrode array. This gave rise to a hiPSC-derived closed-loop cardiac tissue (iCT), enabling spontaneous TW initiation and swift pacing of cardiomyocytes from various cell lines. The TW-paced cardiomyocytes demonstrated heightened sarcomeric and functional maturation, exhibiting enhanced response to isoproterenol. Moreover, these cells showcased diminished sensitivity to verapamil and maintained low arrhythmia rates with ranolazine-two drugs associated with a low risk of torsades de pointes (TdP). Notably, the TW group displayed increased arrhythmia rates with high and intermediate risk TdP drugs (quinidine and pimozide), underscoring the potential utility of this system in drug assessment applications.

Combined administration of laminin-221 and prostacyclin agonist enhances endogenous cardiac repair in an acute infarct rat heart.

Although endogenous cardiac repair by recruitment of stem cells may serve as a therapeutic approach to healing a damaged heart, how to effectively enhance the migration of stem cells to the damaged heart is unclear. Here, we examined whether the combined administration of prostacyclin agonist (ONO1301), a multiple-cytokine inducer, and stem cell niche laminin-221 (LM221), enhances regeneration through endogenous cardiac repair. We administered ONO1301- and LM221-immersed sheets, LM221-immersed sheets, ONO1301-immersed sheets, and PBS-immersed sheets (control) to an acute infarction rat model. Four weeks later, cardiac function, histology, and cytokine expression were analysed. The combined administration of LM221 and ONO1301 upregulated angiogenic and chemotactic factors in the myocardium after 4 weeks and enhanced the accumulation of ILB4 positive cells, SMA positive cells, and platelet-derived growth factor receptor alpha (PDGFRα) and CD90 double-positive cells, leading to the generation of mature microvascular networks. Interstitial fibrosis reduced and functional recovery was prominent in LM221- and ONO1301-administrated hearts as compared with those in ONO1301-administrated or control hearts. LM221 and ONO1301 combination enhanced recruitment of PDGFRα and CD90 double-positive cells, maturation of vessels, and functional recovery in rat acute myocardial infarction hearts, highlighting a new promising acellular approach for the failed heart.

Large-Scale Differentiation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes by Stirring-Type Suspension Culture.

Regenerative medicine using human-induced pluripotent stem cells (hiPSCs) is a promising approach to treat heart failure. However, a large number of cells are required to achieve the desired therapeutic effect. The stirring-type suspension culture method allows a large-scale production of hiPSC-derived cardiomyocytes (more than 1 × 108 cells/100 mL), leading to a stable cell supply. Here, we describe a method to scale-up hiPSC-derived cardiomyocyte production with a high differentiation efficiency.

Pioglitazone strengthen therapeutic effect of adipose-derived regenerative cells against ischemic cardiomyopathy through enhanced expression of adiponectin and modulation of macrophage phenotype.

BACKGROUND: The efficacy of cell transplantation in heart failure is reportedly modest, but adjuvant drugs combined with cell therapy may improve this efficacy. Peroxisome proliferator-activated receptor (PPAR)γ, one of the hypoglycemic medicine for diabetes mellitus, reportedly enhances cytokine production in adipose tissue-derived regenerative cells (ADRCs). We hypothesized that combined administration of PPARγ agonists and ADRCs may enhance the paracrine effects of adiponectin (APN), leading to functional recovery in a chronic myocardial infarction (MI) model. METHODS: ADRCs were isolated from adipose tissues of adult rats by gradient centrifugation and embedded in bio-compatible fibrin-glue to produce ADRCs grafts. In the in vitro study, the ADRCs grafts released APN, which was significantly enhanced by the PPARγ agonist (PGZ, pioglitazone). Transplantation of ADRCs grafts (group A), ADRCs mixed with PGZ (group AP), APN knockdown-ADRCs (group Si) or PGZ (group P) onto the epicardium or a sham operation (group C) was performed (n = 10-20 per group). RESULTS: The AP group showed significant improvement in ejection fraction compared to that in the other groups. In the AP group, a significantly larger number of M2-polarized macrophages was detected and existed for a significantly longer duration in the infarct area. Furthermore, comparing Si group and P group, western blotting of T-cadherin revealed that exogenous APN and local expression of T-cadherin were essential to this histological change and recovery of cardiac function. CONCLUSIONS: Combined administration of PPARγ agonist and ADRSCs activated M2-polarized macrophages with enhancement of APN paracrine effects and lead to better cardiac function in a rat infarction model.

Tumorigenicity assay essential for facilitating safety studies of hiPSC-derived cardiomyocytes for clinical application.

Transplantation of cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSC-CMs) is a promising treatment for heart failure, but residual undifferentiated hiPSCs and malignant transformed cells may lead to tumor formation. Here we describe a highly sensitive tumorigenicity assay for the detection of these cells in hiPSC-CMs. The soft agar colony formation assay and cell growth analysis were unable to detect malignantly transformed cells in hiPSC-CMs. There were no karyotypic abnormalities during hiPSCs subculture and differentiation. The hiPSC markers TRA1-60 and LIN28 showed the highest sensitivity for detecting undifferentiated hiPSCs among primary cardiomyocytes. Transplantation of hiPSC-CMs with a LIN28-positive fraction > 0.33% resulted in tumor formation in nude rats, whereas no tumors were formed when the fraction was < 0.1%. These findings suggested that combination of these in vitro and in vivo tumorigenecity assays can verify the safety of hiPSC-CMs for cell transplantation therapy.

Laminin-511 Supplementation Enhances Stem Cell Localization With Suppression in the Decline of Cardiac Function in Acute Infarct Rats.

BACKGROUND: The extracellular matrix, in particular basement membrane components such as laminins (LMs), is essential for stem cell differentiation and self-renewal. LM511 and LM221 are the main extracellular matrix components of the epicardium, where stem cells were abundant. Here, we examined whether LMs affected the regeneration process by modulating stem cell activities. METHODS: In vitro, adhesive, and proliferative activities of mesenchymal stem cells (MSCs) were evaluated on LM511 and LM221. To examine the effects of LMs in vivo, we established an acute myocardial infarction model by ligation of the proximal part of the left anterior descending artery at the height of the left atrial appendage and then placed atelocollagen sheets with or without LM511 and LM221 over the anterolateral surface of the left ventricular wall. Four or 8 weeks later, cardiac function, histology, and cytokine expressions were analyzed. RESULTS: MSCs showed greater proliferation and adhesive properties on LM511 than on LM221. In vivo, at 4 weeks, isolectin B4-positive cells were significantly higher in the LM511-transplanted group than in the control group. Moreover, some isolectin B4-positive cells expressed both platelet-derived growth factor receptor α and CD90, suggesting that LM511 enhanced MSC recruitment and attachment at the implanted site. After 8 weeks, these cells were more abundant than at 4 weeks. Transplantation with LM511-conjugated sheets increased the expression of cardioprotective and angiogenic factors. CONCLUSIONS: Transplantation with LM511-conjugated sheets enhanced MSC localization to the implantation site and modulated stem cells activities, leading to angiogenesis in acute myocardial infarction rat models.

Immunologic targeting of CD30 eliminates tumourigenic human pluripotent stem cells, allowing safer clinical application of hiPSC-based cell therapy.

Induced pluripotent stem cells (iPSCs) are promising candidate cells for cardiomyogenesis in the failing heart. However, teratoma/tumour formation originating from undifferentiated iPSCs contaminating the graft is a critical concern for clinical application. Here, we hypothesized that brentuximab vedotin, which targets CD30, induces apoptosis in tumourigenic cells, thus increasing the safety of iPSC therapy for heart failure. Flow cytometry analysis identified consistent expression of CD30 in undifferentiated human iPSCs. Addition of brentuximab vedotin in vitro for 72 h efficiently induced cell death in human iPSCs, associated with a significant increase in G2/M phase cells. Brentuximab vedotin significantly reduced Lin28 expression in cardiomyogenically differentiated human iPSCs. Transplantation of human iPSC-derived cardiomyocytes (CMs) without treatment into NOG mice consistently induced teratoma/tumour formation, with a substantial number of Ki-67-positive cells in the graft at 4 months post-transplant, whereas iPSC-derived CMs treated with brentuximab vedotin prior to the transplantation did not show teratoma/tumour formation, which was associated with absence of Ki-67-positive cells in the graft over the same period. These findings suggest that in vitro treatment with brentuximab vedotin, targeting the CD30-positive iPSC fraction, reduced tumourigenicity in human iPSC-derived CMs, potentially providing enhanced safety for iPSC-based cardiomyogenesis therapy in clinical scenarios.

Development of In Vitro Drug-Induced Cardiotoxicity Assay by Using Three-Dimensional Cardiac Tissues Derived from Human Induced Pluripotent Stem Cells.

An in vitro drug-induced cardiotoxicity assay is a critical step in drug discovery for clinical use. The use of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is promising for this purpose. However, single hiPSC-CMs are limited in their ability to mimic native cardiac tissue structurally and functionally, and the generation of artificial cardiac tissue using hiPSC-CMs is an ongoing challenging. We therefore developed a new method of constructing three-dimensional (3D) artificial tissues in a short time by coating extracellular matrix (ECM) components on cell surfaces. We hypothesized that 3D cardiac tissues derived from hiPSC-CMs (3D-hiPSC-CT) could be used for an in vitro drug-induced cardiotoxicity assay. 3D-hiPSC-CT were generated by fibronectin and gelatin nanofilm coated single hiPSC-CMs. Histologically, 3D-hiPSC-CT exhibited a sarcomere structure in the myocytes and ECM proteins, such as fibronectin, collagen type I/III, and laminin. The administration of cytotoxic doxorubicin at 5.0 µM induced the release of lactate dehydrogenase, while that at 2.0 µM reduced the cell viability. E-4031, human ether-a-go-go related gene (hERG)-type potassium channel blocker, and isoproterenol induced significant changes both in the Ca transient parameters and contractile parameters in a dose-dependent manner. The 3D-hiPSC-CT exhibited doxorubicin-sensitive cytotoxicity and hERG channel blocker/isoproterenol-sensitive electrical activity in vitro, indicating its usefulness for drug-induced cardiotoxicity assays or drug screening systems for drug discovery.

Pivotal Role of Non-cardiomyocytes in Electromechanical and Therapeutic Potential of Induced Pluripotent Stem Cell-Derived Engineered Cardiac Tissue.

Although engineered cardiac tissues (ECTs) derived from induced pluripotent stem cells (iPSCs) are promising for myocardial regenerative therapy, the appropriate ratio of cardiomyocytes to non-cardiomyocytes is not fully understood. Here, we determined whether ECT-cell content is a key determinant of its structure/function, thereby affecting ECT therapeutic potential for advanced heart failure. Scaffold-free ECTs containing different ratios (25%, 50%, 70%, or 90%) of iPSC-derived cardiomyocytes were generated by magnetic-activated cell sorting by using cardiac-specific markers. Notably, ECTs showed synchronized spontaneous beating when cardiomyocytes constituted ≥50% of total cells, with the electrical-conduction velocity increasing depending on cardiomyocyte ratio; however, ECTs containing 90% cardiomyocytes failed to form stable structures. ECTs containing 25% or 50% cardiomyocytes predominantly expressed collagen and fibronectin, whereas ECTs containing 70% cardiomyocytes predominantly expressed laminin and exhibited the highest contractile/relaxation properties. Furthermore, transplantation of ECTs containing 50% or 70% cardiomyocytes into a rat chronic myocardial infarction model led to a more profound functional recovery as compared with controls. Notably, transplanted ECTs showed electrical synchronization with the native heart under Langendorff perfusion. Collectively, these results indicate that the quantity of non-cardiomyocytes is critical in generating functional iPSC-derived ECTs as grafts for cardiac-regeneration therapy, with ECTs containing 50-70% cardiomyocytes exhibiting stable structures and increased cardiotherapeutic potential.

Development of a practical sandwich assay to detect human pluripotent stem cells using cell culture media.

Human pluripotent stem cells are considered to be ideal cell sources for regenerative medicine, but their clinical and industrial application is hindered by their tumorigenic potential. Previously we have identified a pluripotent stem cell-specific lectin rBC2LCN recognizing podocalyxin as a cell surface ligand. More recently, podocalyxin was found to be a soluble ligand of rBC2LCN that is secreted specifically from human pluripotent stem cells into cell culture media. Taking advantage of this phenomenon, we have previously developed a sandwich assay targeting the soluble podocalyxin using rBC2LCN as a capturing probe and another lectin rABA as an overlay probe to detect human pluripotent stem cells residing in cell therapy products derived from human pluripotent stem cells. A drawback to this, however, was that cell culture media containing fetal bovine serum was found to cause a substantial background signal to the sandwich assay. To reduce the background and increase the sensitivity, we screened different overlay probes to detect the soluble podocalyxin. Among them, an anti-keratan sulfate monoclonal antibody called R-10G showed the highest sensitivity and provided a low background signal to fetal bovine serum. The established sandwich assay using rBC2LCN and R-10G was proved to be powerful, which allowed the high-sensitive detection of human induced pluripotent stem cells residing among clinical-grade cardiomyocytes and neural stem cells, both derived from human induced pluripotent stem cells. The developed method has a possibility to be a standard technology to detect human induced pluripotent stem cells resided in various types of cell therapy products.

Novel Hyaluronan Formulation Enhances the Efficacy of Boron Neutron Capture Therapy for Murine Mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a refractory cancer of the pleura caused by asbestos exposure. MPM is difficult to treat because it easily disseminates. Boron neutron capture therapy (BNCT) is a radiotherapy in which cancer cells that selectively take up (10)Boron-containing compounds are destroyed, and normal cells are uninjured. Hyaluronan (HA) is a ligand of cluster of differentiation 44 (CD44), that is expressed on MPM cells. MATERIALS AND METHODS: In order to enhance BNCT for MPM tumors, we developed a novel HA-containing (10)B (sodium borocaptate: BSH) formulation (HA-BND-S). We examined the efficacy of HA-BND-S using MPM cells and a mouse MPM model. RESULTS: HA-BND-S preferentially bound MPM cells dose-dependently, and increased the cytotoxicity of BNCT compared to BSH in vitro. HA-BND-S administration significantly increased the survival of MPM tumor-bearing mice compared to BSH at the same (10)B dosage in BNCT. CONCLUSION: Modifying BSH with HA is a promising strategy for enhancing the efficacy of BNCT for therapy of MPM.

Enhanced survival of transplanted human induced pluripotent stem cell-derived cardiomyocytes by the combination of cell sheets with the pedicled omental flap technique in a porcine heart.

BACKGROUND: Transplantation of cardiomyocytes that are derived from human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) shows promise in generating new functional myocardium in situ, whereas the survival and functionality of the transplanted cells are critical in considering this therapeutic impact. Cell-sheet method has been used to transplant many functional cells; however, potential ischemia might limit cell survival. The omentum, which is known to have rich vasculature, is expected to be a source of blood supply. We hypothesized that transplantation of hiPS-CM cell sheets combined with an omentum flap may deliver a large number of functional hiPS-CMs with enhanced blood supply. METHODS AND RESULTS: Retrovirally established human iPS cells were treated with Wnt signaling molecules to induce cardiomyogenic differentiation, followed by superparamagnetic iron oxide labeling. Cell sheets were created from the magnetically labeled hiPS-CMs using temperature-responsive dishes and transplanted to porcine hearts with or without the omentum flap (n=8 each). Two months after transplantation, the survival of superparamagnetic iron oxide-labeled hiPS-CMs, assessed by MRI, was significantly greater in mini-pigs with the omentum than in those without it; histologically, vascular density in the transplanted area was significantly greater in mini-pigs with the omentum than in those without it. The transplanted tissues contained abundant cardiac troponin T-positive cells surrounded by vascular-rich structures. CONCLUSIONS: The omentum flap enhanced the survival of hiPS-CMs after transplantation via increased angiogenesis, suggesting that this strategy is useful in clinical settings. The combination of hiPS-CMs and the omentum flap may be a promising technique for the development of tissue-engineered vascular-rich new myocardium in vivo.

Sustained-release delivery of prostacyclin analogue enhances bone marrow-cell recruitment and yields functional benefits for acute myocardial infarction in mice.

BACKGROUND: A prostacyclin analogue, ONO-1301, is reported to upregulate beneficial proteins, including stromal cell derived factor-1 (SDF-1). We hypothesized that the sustained-release delivery of ONO-1301 would enhance SDF-1 expression in the acute myocardial infarction (MI) heart and induce bone marrow cells (BMCs) to home to the myocardium, leading to improved cardiac function in mice. METHODS AND RESULTS: ONO-1301 significantly upregulated SDF-1 secretion by fibroblasts. BMC migration was greater to ONO-1301-stimulated than unstimulated conditioned medium. This increase was diminished by treating the BMCs with a CXCR4-neutralizing antibody or CXCR4 antagonist (AMD3100). Atelocollagen sheets containing a sustained-release form of ONO-1301 (n = 33) or ONO-1301-free vehicle (n = 48) were implanted on the left ventricular (LV) anterior wall immediately after permanent left-anterior descending artery occlusion in C57BL6/N mice (male, 8-weeks-old). The SDF-1 expression in the infarct border zone was significantly elevated for 1 month in the ONO-1301-treated group. BMC accumulation in the infarcted hearts, detected by in vivo imaging after intravenous injection of labeled BMCs, was enhanced in the ONO-1301-treated hearts. This increase was inhibited by AMD3100. The accumulated BMCs differentiated into capillary structures. The survival rates and cardiac function were significantly improved in the ONO-1301-treated group (fractional area change 23±1%; n = 22) compared to the vehicle group (19±1%; n = 20; P = 0.004). LV anterior wall thinning, expansion of infarction, and fibrosis were lower in the ONO-1301-treated group. CONCLUSIONS: Sustained-release delivery of ONO-1301 promoted BMC recruitment to the acute MI heart via SDF-1/CXCR4 signaling and restored cardiac performance, suggesting a novel mechanism for ONO-1301-mediated acute-MI heart repair.

HDL/apolipoprotein A-I binds to macrophage-derived progranulin and suppresses its conversion into proinflammatory granulins.

AIM: HDL has anti-inflammatory effects on macrophages, although the mechanism of action remains unclear. We hypothesized that HDL suppresses the conversion of macrophage-secreted factors into proinflammatory factors via binding, and tried to identify the factor that could form a complex with HDL and/or apolipoprotein (apo) A-I. METHODS AND RESULTS: In conditioned media obtained from human monocyte-derived macrophages, we found an apo A-I binding protein and identified the protein as progranulin/proepithelin/acrogranin/PCDGF. Co-immunoprecipitation analysis showing that progranulin binds and forms a complex with apo A-I and the presence of progranulin in the HDL fraction in the sera indicated that progranilin is a novel apolipoprotein. Conditioned media of HEK293 cells transfected with progranulin augmented the expression of TNF-alpha and IL-1-beta on macrophages, but these effects of progranulin were inhibited by co-incubation with HDL or apo A-I. Anti-progranulin antibodies also reduced the expression of TNF-alpha and IL-1-beta on macrophages. Granulins as conversion products derived from progranilin increased TNF-alpha and IL-1-beta expression and the effects were not suppressed by HDL. CONCLUSIONS: Our results suggest that the anti-inflammatory effects of HDL on macrophages might be due to suppression of the conversion of progranulin into proinflammatory granulins by forming a complex.

Reduction of N-glycolylneuraminic acid xenoantigen on human adipose tissue-derived stromal cells/mesenchymal stem cells leads to safer and more useful cell sources for various stem cell therapies.

Adipose tissue is an attractive source for somatic stem cell therapy. Currently, human adipose tissue-derived stromal cells/mesenchymal stem cells (hADSCs/MSCs) are cultured with fetal bovine serum (FBS). Recently, however, not only human embryonic stem cell lines cultured on mouse feeder cells but also bone marrow-derived human MSCs cultured with FBS were reported to express N-glycolylneuraminic acid (Neu5Gc) xenoantigen. Human serum contains high titers of natural preformed antibodies against Neu5Gc. We studied the presence of Neu5Gc on hADSCs/MSCs cultured with FBS and human immune response mediated by Neu5Gc. Our data indicated that hADSCs/MSCs cultured with FBS expressed Neu5Gc and that human natural preformed antibodies could bind to hADSCs/MSCs. However, hADSCs/MSCs express complement regulatory proteins such as CD46, CD55, and CD59 and are largely resistant to complement-mediated cytotoxicity. hADSCs/MSCs cultured with FBS could be injured by antibody-dependent cell-mediated cytotoxicity mechanism. Further, human monocyte-derived macrophages could phagocytose hADSCs/MSCs cultured with FBS and this phagocytic activity was increased in the presence of human serum. Culturing hADSCs/MSCs with heat-inactivated human serum for a week could markedly reduce Neu5Gc on hADSCs/MSCs and prevent immune responses mediated by Neu5Gc, such as binding of human natural preformed antibodies, antibody-dependent cell-mediated cytotoxicity, and phagocytosis. Adipogenic and osteogenic differentiation potentials of hADSCs/MSCs cultured with heat-inactivated human serum were not less than that of those cultured with FBS. For stem cell therapies based on hADSCs/MSCs, hADSCs/MSCs that presented Neu5Gc on their cell surfaces after exposure to FBS should be cleaned up to be rescued from xenogeneic rejection.