CT-001, a novel fast-clearing factor VIIa, enhanced the hemostatic activity in postpartum samples.

ABSTRACT: The hemostatic system is upregulated to protect pregnant mothers from hemorrhage during childbirth. Studies of the details just before and after delivery, however, are lacking. Recombinant factor VIIa (rFVIIa) has recently been granted approval by the European Medicines Agency for the treatment of postpartum hemorrhage (PPH). A next-generation molecule, CT-001, is being developed as a potentially safer and more efficacious rFVIIa-based therapy. We sought to evaluate the peripartum hemostatic status of pregnant women and assess the ex vivo hemostatic activity of rFVIIa and CT-001 in peripartum blood samples. Pregnant women from 2 study sites were enrolled in this prospective observational study. Baseline blood samples were collected up to 3 days before delivery. Postdelivery samples were collected 45 (±15) minutes after delivery. Between the 2 time points, soluble fibrin monomer and D-dimer increased whereas tissue factor, FVIII, FV, and fibrinogen decreased. Interestingly, the postdelivery lag time and time to peak in the thrombin generation assay were shortened, and the peak thrombin generation capacity was maintained despite the reduced levels of coagulation proteins after delivery. Furthermore, both rFVIIa and CT-001 were effective in enhancing clotting activity of postdelivery samples in activated partial thromboplastin time, prothrombin time, thrombin generation, and viscoelastic hemostatic assays, with CT-001 demonstrating greater activity. In conclusion, despite apparent ongoing consumption of coagulation factors at the time of delivery, thrombin output was maintained. Both rFVIIa and CT-001 enhanced the upregulated hemostatic activity in postdelivery samples, and consistent with previous studies comparing CT-001 and rFVIIa in vitro and in in vivo, CT-001 demonstrated greater activity than rFVIIa.

Modification of a purification and expansion method for human embryonic stem cell-derived cardiomyocytes.

OBJECTIVE: This study aimed to develop a simple and efficient purification method for human embryonic stem cell (hESC)-derived cardiomyocytes (CMs) using a low-glucose culture system. In addition, we investigated whether intercellular adhesion between single hESC-CMs plays a critical role in enhancing proliferation of purified hESC-CMs. METHOD: hESCs were cultured in suspension to form human embryoid bodies (hEBs) from which ∼15% contracting clusters were derived after 15-20 days in culture. To purify CMs from contracting hEBs, we first manually isolated contracting clumps that were re-cultured on gelatin-coated plates with media containing a low concentration of glucose. The purified hESC-CMs were cultured at different densities to examine whether cell-cell contact enhances proliferation of hESC-CMs. RESULTS: Purified CMs demonstrated spontaneous contraction and strongly expressed the CM-specific markers cardiac troponin T and slow myosin heavy chain. We investigated the purification efficiency by examining the expression levels of cardiac-related genes and the expression of MitoTracker Red dye. In addition, purified hESC-CMs in low-glucose culture demonstrated a 1.5-fold increase in their proliferative capacity compared to those cultured as single hESC-CMs. CONCLUSION: A low level of glucose is efficient in purifying hESC-CMs and intercellular adhesion between individual hESC-CMs plays a critical role in enhancing hESC-CM proliferation.

From Bench to Market: Preparing Human Pluripotent Stem Cells Derived Cardiomyocytes for Various Applications.

Human cardiomyocytes (CMs) cease to proliferate and remain terminally differentiated thereafter, when humans reach the mid-20s. Thus, any damages sustained by myocardium tissue are irreversible, and they require medical interventions to regain functionality. To date, new surgical procedures and drugs have been developed, albeit with limited success, to treat various heart diseases including myocardial infarction. Hence, there is a pressing need to develop more effective treatment methods to address the increasing mortality rate of the heart diseases. Functional CMs are not only an important in vitro cellular tool to model various types of heart diseases for drug development, but they are also a promising therapeutic agent for cell therapy. However, the limited proliferative capacity entails difficulties in acquiring functional CMs in the scale that is required for pathological studies and cell therapy development. Stem cells, human pluripotent stem cells (hPSCs) in particular, have been considered as an unlimited cellular source for providing functional CMs for various applications. Notable progress has already been made: the first clinical trials of hPSCs derived CMs (hPSC-CMs) for treating myocardial infarction was approved in 2015, and their potential use in disease modeling and drug discovery is being fully explored. This concise review gives an account of current development of differentiation, purification and maturation techniques for hPSC-CMs, and their application in cell therapy development and pharmaceutical industries will be discussed with the latest experimental evidence.

Metabolome Profiling of Partial and Fully Reprogrammed Induced Pluripotent Stem Cells.

Acquisition of proper metabolomic fate is required to convert somatic cells toward fully reprogrammed pluripotent stem cells. The majority of induced pluripotent stem cells (iPSCs) are partially reprogrammed and have a transcriptome different from that of the pluripotent stem cells. The metabolomic profile and mitochondrial metabolic functions required to achieve full reprogramming of somatic cells to iPSC status have not yet been elucidated. Clarification of the metabolites underlying reprogramming mechanisms should enable further optimization to enhance the efficiency of obtaining fully reprogrammed iPSCs. In this study, we characterized the metabolites of human fully reprogrammed iPSCs, partially reprogrammed iPSCs, and embryonic stem cells (ESCs). Using capillary electrophoresis time-of-flight mass spectrometry-based metabolomics, we found that 89% of analyzed metabolites were similarly expressed in fully reprogrammed iPSCs and human ESCs (hESCs), whereas partially reprogrammed iPSCs shared only 74% similarly expressed metabolites with hESCs. Metabolomic profiling analysis suggested that converting mitochondrial respiration to glycolytic flux is critical for reprogramming of somatic cells into fully reprogrammed iPSCs. This characterization of metabolic reprogramming in iPSCs may enable the development of new reprogramming parameters for enhancing the generation of fully reprogrammed human iPSCs.

Perivascular Progenitor Cells Derived From Human Embryonic Stem Cells Exhibit Functional Characteristics of Pericytes and Improve the Retinal Vasculature in a Rodent Model of Diabetic Retinopathy.

UNLABELLED: : Diabetic retinopathy (DR) is the leading cause of blindness in working-age people. Pericyte loss is one of the pathologic cellular events in DR, which weakens the retinal microvessels. Damage to the microvascular networks is irreversible and permanent; thus further progression of DR is inevitable. In this study, we hypothesize that multipotent perivascular progenitor cells derived from human embryonic stem cells (hESC-PVPCs) improve the damaged retinal vasculature in the streptozotocin-induced diabetic rodent models. We describe a highly efficient and feasible protocol to derive such cells with a natural selection method without cell-sorting processes. As a cellular model of pericytes, hESC-PVPCs exhibited marker expressions such as CD140B, CD146, NG2, and functional characteristics of pericytes. Following a single intravitreal injection into diabetic Brown Norway rats, we demonstrate that the cells localized alongside typical perivascular regions of the retinal vasculature and stabilized the blood-retinal barrier breakdown. Findings in this study highlight a therapeutic potential of hESC-PVPCs in DR by mimicking the role of pericytes in vascular stabilization. SIGNIFICANCE: This study provides a simple and feasible method to generate perivascular progenitor cells from human embryonic stem cells. These cells share functional characteristics with pericytes, which are irreversibly lost at the onset of diabetic retinopathy. Animal studies demonstrated that replenishing the damaged pericytes with perivascular progenitor cells could restore retinal vascular integrity and prevent fluid leakage. This provides promising and compelling evidence that perivascular progenitor cells can be used as a novel therapeutic agent to treat diabetic retinopathy patients.

A porous membrane-mediated isolation of mesenchymal stem cells from human embryonic stem cells.

Pluripotent human embryonic stem cells (hESCs) acquire mesenchymal characteristics during the epithelial-mesenchymal transition (EMT) process. Here, we report a simple and an efficient isolation method for mesenchymal stem cells (MSCs) from hESCs undergoing EMT using a commercialized porous membrane transwell culture insert. Suspension culture of hESC colonies results in the formation of embryoid bodies, which adhered on the upper compartment of 8 µm porous membrane in the presence of EMG2-MV media. The population migrating through the permeable membrane to the lower compartment not only exhibited EMT markers but also expressed high levels of a panel of typical MSC surface antigen markers, and demonstrated multipotent differentiation capability. In addition, they have a prolonged proliferation capacity without characteristics and chromosomal changes. Furthermore, the isolated MSCs significantly enhanced cardiac functions in a rat model of myocardial infarction (MI) as measured by the left ventricle wall thickness (MI control, 32.9%±3.2% vs. hESCs-MSCs, 38.7%±2.4%), scar length (MI control, 46.1%±2.5% vs. hESCs-MSCs, 41.8%±1.3%), fibrosis area (MI control, 34.3%±1.6% vs. hESCs-MSCs, 28.9%±3.5%), and capillary density. Our findings demonstrate an ease with which hESCs-MSCs can be effectively isolated using the porous membrane, which overcomes the lack of availability of MSCs for therapeutic applications in various diseased animal models.

Optimizing human embryonic stem cells differentiation efficiency by screening size-tunable homogenous embryoid bodies.

Human embryonic stem cells (hESCs) are generally induced to differentiate by forming spherical structures termed embryoid bodies (EBs) in the presence of soluble growth factors. hEBs are generated by suspending small clumps of hESC colonies; however, the resulting hEBs are heterogeneous because this method lacks the ability to control the number of cells in individual EBs. This heterogeneity affects factors that influence differentiation such as cell-cell contact and the diffusion of soluble factors, and consequently, the differentiation capacity of each EB varies. Here, we fabricated size-tunable concave microwells to control the physical environment, thereby regulating the size of EBs formed from single hESCs. Defined numbers of single hESCs were forced to aggregate and generate uniformly sized EBs with high fidelity, and the size of the EBs was controlled using concave microwells of different diameters. Differentiation patterns in H9- and CHA15-hESCs were affected by EB size in both the absence and presence of growth factors. By screening EB size in the presence of various BMP4 concentrations, a two-fold increase in endothelial cell differentiation was achieved. Because each hESC line has unique characteristics, the findings of this study demonstrate that concave microwells could be used to screen different EB sizes and growth factor concentrations to optimize differentiation for each hESC line.

Nanotopographical control for maintaining undifferentiated human embryonic stem cell colonies in feeder free conditions.

Recently emerging evidence has indicated surface nanotopography as an important physical parameter in the stem cell niche for regulating cell fate and behaviors for various types of stem cells. In this study, a substrate featuring arrays of increasing nanopillar diameter was devised to investigate the effects of varying surface nanotopography on the maintenance of undifferentiated human embryonic stem cells (hESC) colonies in the absence of feeder cells. Single hESCs cultured across gradient nanopattern (G-Np) substrate were generally organized into compact colonies, and expressed higher levels of undifferentiated markers compared to those cultured on the unstructured control substrate. In particular, hESC demonstrates a propensity to organize into more compact colonies expressing higher levels of undifferentiated markers towards a smaller nanopillar diameter range (D = 120-170 nm). Cell-nanotopography interactions modulated the formation of focal adhesions and cytoskeleton reorganization to restrict colony spreading, which reinforced E-cadherin mediated cell-cell adhesions in hESC colonies. Maintaining compact hESC colony integrity revealed to be indispensable for hESC undifferentiated state as the loss of cell-cell adhesions within spreading hESC on the control substrate exhibited morphological and gene expression signatures of epithelial-to-mesenchymal transition-like processes. Findings in this study demonstrated a feasible approach to screen the optimal nanotopographical cues for maintaining undifferentiated hESC colonies in feeder free conditions, which provides a platform for further investigations into developing hESC feeder free culture systems for the purpose of regenerative medicine.

Development of a xeno-free autologous culture system for endothelial progenitor cells derived from human umbilical cord blood.

Despite promising preclinical outcomes in animal models, a number of challenges remain for human clinical use. In particular, expanding a large number of endothelial progenitor cells (EPCs) in vitro in the absence of animal-derived products is the most critical hurdle remaining to be overcome to ensure the safety and efficiency of human therapy. To develop in vitro culture conditions for EPCs derived from human cord blood (hCB-EPCs), we isolated extracts (UCE) and collagen (UC-collagen) from umbilical cord tissue to replace their animal-derived counterparts. UC-collagen and UCE efficiently supported the attachment and proliferation of hCB-EPCs in a manner comparable to that of animal-derived collagen in the conventional culture system. Our developed autologous culture system maintained the typical characteristics of hCB-EPCs, as represented by the expression of EPC-associated surface markers. In addition, the therapeutic potential of hCB-EPCs was confirmed when the transplantation of hCB-EPCs cultured in this autologous culture system promoted limb salvage in a mouse model of hindlimb ischemia and was shown to contribute to attenuating muscle degeneration and fibrosis. We suggest that the umbilical cord represents a source for autologous biomaterials for the in vitro culture of hCB-EPCs. The main characteristics and therapeutic potential of hCB-EPCs were not compromised in developed autologous culture system. The absence of animal-derived products in our newly developed in vitro culture removes concerns associated with secondary contamination. Thus, we hope that this culture system accelerates the realization of therapeutic applications of autologous hCB-EPCs for human vascular diseases.

The use of aggregates of purified cardiomyocytes derived from human ESCs for functional engraftment after myocardial infarction.

Embryonic stem cells (ESCs) have the capacity to undergo directed differentiation into contracting cardiomyocytes. Therefore, functional cardiomyocytes derived from human embryonic stem cells (hESC-CMs) are potential candidates for cellular cardiomyoplasty to regenerate the myocardium after infarction. However, the directed differentiation of hESCs induces not only contracting cardiomyocytes but also other cell types. Thus, a risk of teratoma formation and oncologic transformation exists following the transplantation of hESC-CMs containing other cell lineages. In addition, the transplantation of hESC-CMs into the infarcted myocardium limits therapeutic efficacy due to low viability and poor engraftment. In this study, we established an efficient preparation method to obtain pure contracting cardiomyocytes from hESCs. We also developed a delivery system to achieve enhanced viability and a functional connection with the host myocardium after transplantation in a myocardial infarction model. A serum-free medium was used to obtain pure contracting cardiomyocytes from other cell lineages after the cardiac differentiation of hESCs. Aggregates of purified hESC-CMs were formed, and then the expression of cardiomyocyte-specific markers and the viability of the aggregated CMs were examined in hypoxic conditions. In addition, we determined whether the viability of the hESC-CMs and their ability to engraft with the host myocardium could be enhanced by transplanting them as aggregates in a myocardial infarction model. The therapeutic efficacy of the cardiomyocytes was examined by immunohistochemical analyses as well as physiological analyses of left-ventricular function. We found that the transplantation of contracting hESC-CM aggregates improved their survival and function in infarcted rat hearts in comparison to the transplantation of dissociated cells. Our method using hESC-CMs can be considered an effective strategy for clinical applications without critical barriers.

Hypoxia enhances the generation of retinal progenitor cells from human induced pluripotent and embryonic stem cells.

The efficient differentiation of retinal cells from human pluripotent stem cells remains a major challenge for the development of successful and cost-effective cellular therapies for various forms of blindness. Current differentiation strategies rely on exposing pluripotent stem cells to soluble growth factors that play key roles during early development (such as DKK-1, Noggin, and IGF-1) at 20% oxygen (O(2)). This O(2) tension is, however, considerably higher than O(2) levels during organogenesis and may impair the differentiation process. In this study, we examined the effect of mimicking the physiological O(2) tension (2%) on the generation of retinal progenitor cells (RPCs) from human induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs). Both cell types were induced to differentiate into RPCs at 20% and 2% O(2). After 3 days in suspension culture as embryoid bodies (EBs), 2% O(2) caused the activation of hypoxia inducible factor responsive genes VEGF and LDHA and was accompanied by elevated expression levels of the early eye field genes Six3 and Lhx2. Twenty-one days after plating EBs in an adherent culture, we observed more RPCs co-expressing Pax6 and Chx10 at 2% O(2). Quantitative polymerase chain reaction analysis confirmed that lowering O(2) tension had caused a rise in the expression of both genes compared with 20% O(2). Our results indicate that mimicking physiological O(2) is a favorable condition for the efficient generation of RPCs from both hiPSCs and hESCs.