Human embryonic stem cells: possibilities for human cell transplantation

Human embryonic stem (ES) cells serve as a potentially unlimited renewable source for cell transplantation targeted to treat several diseases. One advantage of embryonic stem (ES) cells over other stem cells under research is their apparently indefinite self-renewal capacity if cultured appropriately, and their ready differentiation into various cell phenotypes of all three germ layers. To date, a number of studies have reported the derivation of specific functional derivatives from human ES cells in vitro. While there have been clinical trials of human embryonal carcinoma (EC) cell-derived neurons in humans there has been no attempt as yet using human ES cell derivatives. However, the latter have been transplanted into recipient animals. In some cases ES-derived cells were shown to undergo further maturation, displayed integration with host tissue and even ameliorated the disease condition in the animal model. Recently, it has been reported that human ES cells can be genetically manipulated. Such procedures could be used to direct differentiation to a specific cell type or to reduce graft rejections by the modification of immune responses. This review highlights some of the recent advances in the field and the challenges that lie ahead before clinical trials using ES-derived cells can be contemplated.     

Differential bone‐forming capacity of osteogenic cells from either embryonic stem cells or bone marrow‐derived mesenchymal stem cells

For more than a decade, human mesenchymal stem cells (hMSCs) have been used in bone tissue‐engineering research. More recently some of the focus in this field has shifted towards the use of embryonic stem cells. While it is well known that hMSCs are able to form bone when implanted subcutaneously in immune‐deficient mice, the osteogenic potential of embryonic stem cells has been mainly assessed in vitro. Therefore, we performed a series of studies to compare the in vitro and in vivo osteogenic capacities of human and mouse embryonic stem cells to those of hMSCs. Embryonic and mesenchymal stem cells showed all characteristic signs of osteogenic differentiation in vitro when cultured in osteogenic medium, including the deposition of a mineralized matrix and expression of genes involved in osteogenic differentiation. As such, based on the in vitro results, osteogenic ES cells could not be discriminated from osteogenic hMSCs. Nevertheless, although osteogenic hMSCs formed bone upon implantation, osteogenic cells derived from both human and mouse embryonic stem cells did not form functional bone, indicated by absence of osteocytes, bone marrow and lamellar bone. Although embryonic stem cells show all signs of osteogenic differentiation in vitro, it appears that, in contrast to mesenchymal stem cells, they do not possess the ability to form bone in vivo when a similar culture method and osteogenic differentiation protocol was applied. Copyright © 2010 John Wiley & Sons, Ltd.     

Stem cells and nuclear reprogramming

Derivation of human embryonic stem (ES) cells from preimplantation embryos ten years ago raised great hopes that they may be an excellent source of cells for cell replacement therapy. However, serious ethical concerns and the risk of immune rejection of allotransplanted cells have hindered the translation of ES cell‐based therapies into the clinic. In an attempt to circumvent these barriers, a number of methods have been developed for converting adult somatic cells into a pluripotent state from which ethically acceptable patient‐specific mature cells of interest could be derived. These efforts, backed by advances in elucidating the molecular basis of pluripotency, have culminated in successful reprogramming of fibroblasts into ES cell‐like cells, termed induced pluripotent stem (iPS) cells, by ectopic expression of only a handful of “stemness” factors. iPS cells possess morphological, molecular and developmental features of conventional blastocyst‐derived ES cells and have the potential to serve as a source of therapeutic cells for customized tissue repair, gene therapy, drug discovery, toxicological testing and for studying the molecular basis of human disease. The goal of this review is to provide the current state‐of‐the‐art in this very exciting and dynamic field and to discuss barriers that remain to be removed before the therapeutic potential of iPS cells can be fully realized.     

Embryonic stem cells: new possible therapy of degenerative diseases

The capacity of embryonic stem (ES) cells for virtually unlimited self-renewal and differentiation has opened up the prospect of widespread applications in biomedical research and regenerative medicine. The use of these cells would allow to overcome the problems of donor tissue shortage and also implant rejection if the cells are made immunocompatible with the recipient. Since the derivation in 1998 of human ES cell lines from pre-implantation embryos, considerable research is centered on their biology, on how differentiation can be encouraged towards particular cell lineages and also on means to enrich and purify derivative cell types. In addition, ES cells may be used as an in vitro system not only to study cell differentiation but also to evaluate the effects of new drugs and the identification of genes as potential therapeutic targets. This review will summarize what is known about animal and human ES cells with particular emphasis on their application in four animal models of human diseases. Present studies of mouse ES cell transplantation reveal encouraging results but also technical barriers that have to be overcome before clinical trials can be considered.     

Tyrosine kinase signalling in embryonic stem cells

Pluripotent ES (embryonic stem) cells can be expanded in culture and induced to differentiate into a wide range of cell types. Self-renewal of ES cells involves proliferation with concomitant suppression of differentiation. Some critical and conserved pathways regulating self-renewal in both human and mouse ES cells have been identified, but there is also evidence suggesting significant species differences. Cytoplasmic and receptor tyrosine kinases play important roles in proliferation, survival, self-renewal and differentiation in stem, progenitor and adult cells. The present review focuses on the role of tyrosine kinase signalling for maintenance of the undifferentiated state, proliferation, survival and early differentiation of ES cells.     

Stem cell‐based therapy for Parkinson's disease

Motor dysfunctions in Parkinson's disease are considered to be primarily due to the degeneration of dopaminergic neurons in the substantia nigra pars compacta. Pharmacological therapies based on the principle of dopamine replacement are extremely valuable, but suffer from two main drawbacks: troubling side effects (e.g. dyskinesia) and loss of efficacy with disease progression. Transplantation of embryonic dopaminergic neurons has emerged as a therapeutic alternative. Enthusiasm following the success of the initial open‐label trials has been dampened by the negative outcome of double‐blind placebo controlled trials. Additionally, the emergence of graft‐related dyskinesia indicates that the experimental grafting procedure requires further refinement before it can be developed into a therapy. Shortage of embryonic donor tissue limits large‐scale clinical transplantation trials. We review three of the most attractive tissue sources of dopaminergic neurons for cell replacement therapy: human embryonic ventral mesencephalic tissue, embryonic and adult multipotent region‐specific stem cells and embryonic stem cells. Recent developments in embryonic stem cell research and on their implications for a future transplantation therapy in Parkinson's disease are described. Finally, we discuss how human embryonic stem cells can be differentiated into dopaminergic neurons, and issues such as the numbers of dopaminergic neurons required for success and the risk for teratoma formation after implantation.     

Generation, culture, and differentiation of human embryonic stem cells for therapeutic applications.

Embryonic stem (ES) cells, derived from the inner cell mass of the mammalian blastocyst, can continuously proliferate in an undifferentiated state and can also be induced to differentiate into a desired cell lineage. These abilities make ES cells an appealing source for cell replacement therapies, the study of developmental biology, and drug/toxin screening studies. As compared to mouse ES cells, human ES cells have only recently been derived and studied. Although there are many differences in properties between mouse and human ES cells, the study of mouse ES cells has provided important insights into human ES cell research. In this review, we describe the advantages and disadvantages of methods used for human ES cell derivation, the expansion of human ES cells, and the current status of human ES cell differentiation research. In addition, we discuss the endeavor that scientists have undertaken toward the therapeutic application of these cells, which includes therapeutic cloning and the improvement of human ES cell culture conditions.     

Cardiomyocytes from embryonic stem cells: towards human therapy

Background: Despite improvements in survival with current therapies, it is now clear that major cardiovascular event rates in patients remain high. Cardiac cell replacement therapy by using human embryonic stem cell-derived cardiomyocytes has emerged as a promising future approach to regenerate functional myocardium. However, there are still many hurdles to be overcome for the clinical application of these cells. Objective: This review describes the embryonic stem cell system, their differentiation into cardiomyocytes, and functional characterization of the cardiac lineage derivatives in culture and upon in vivo engraftment in animal models. Results/conclusion: A better understanding of the characteristics of the cardiomyocytes from human embryonic stem cells not only predicts their behaviour after implantation but will also help in the design of future strategies for cardiac regeneration in vivo.     

The human embryonic stem cell-derived cardiomyocyte as a pharmacological model

Embryonic stem (ES) cells are specialised cells derived from the early embryo, which are capable of both sustained propagation in the undifferentiated state as well as subsequent differentiation into the majority of cell lineages. Human ES cells are being developed for clinical tissue repair, but a number of problems must be addressed before this becomes a reality. However, they also have potential for translational benefit through its use as a test system for screening pharmaceutical compounds. In the cardiac field, present model systems are not ideal for either screening or basic pharmacological/physiological studies. Cardiomyocytes produced from human ES differentiation have advantages for these purposes over the primary isolated cells or the small number of cell lines available. This review describes the methodology for obtaining cardiomyocytes from human embryonic stem cell-derived cardiomyocyte (hESCM), for increasing the proportion of cardiomyocytes in the preparation and for isolating single embryonic stem cell-derived cardiomyocyte (ESCM) from clusters. Their morphological, contractile and electrophysiological characteristics are compared to mature and immature primary cardiomyocytes. The advantages and disadvantages of the hESCM preparation for long term culture and genetic manipulation are described. Basic pharmacological studies on adrenoceptors and muscarinic receptors in hESCM have been performed, and have given stable and reproducible responses. Prolongation of repolarisation can be detected using hESCM cultured on multielectrode arrays (MEA). Human ESCM have a clear potential to improve model systems available for both basic scientific studies and pharmaceutical screening of cardiac target compounds.     

Recurrent genomic instability of chromosome 1q in neural derivatives of human embryonic stem cells

Human pluripotent stem cells offer a limitless source of cells for regenerative medicine. Neural derivatives of human embryonic stem cells (hESCs) are currently being used for cell therapy in 3 clinical trials. However, hESCs are prone to genomic instability, which could limit their clinical utility. Here, we report that neural differentiation of hESCs systematically produced a neural stem cell population that could be propagated for more than 50 passages without entering senescence; this was true for all 6 hESC lines tested. The apparent spontaneous loss of evolution toward normal senescence of somatic cells was associated with a jumping translocation of chromosome 1q. This chromosomal defect has previously been associated with hematologic malignancies and pediatric brain tumors with poor clinical outcome. Neural stem cells carrying the 1q defect implanted into the brains of rats failed to integrate and expand, whereas normal cells engrafted. Our results call for additional quality controls to be implemented to ensure genomic integrity not only of undifferentiated pluripotent stem cells, but also of hESC derivatives that form cell therapy end products, particularly neural lines.     

Culture conditions have an impact on the maturation of traceable,transplantable mouse embryonic stem cell‐derived otic progenitor cells

The generation of replacement inner ear hair cells (HCs) remains a challenge and stem cell therapy holds the potential for developing therapeutic solutions to hearing and balance disorders. Recent developments have made significant strides in producing mouse otic progenitors using cell culture techniques to initiate HC differentiation. However, no consensus has been reached as to efficiency and therefore current methods remain unsatisfactory. In order to address these issues, we compare the generation of otic and HC progenitors from embryonic stem (ES) cells in two cell culture systems: suspension vs. adherent conditions. In the present study, an ES cell line derived from an Atoh1‐green fluorescent protein (GFP) transgenic mouse was used to track the generation of otic progenitors, initial HCs and to compare these two differentiation systems. We used a two‐step short‐term differentiation method involving an induction period of 5 days during which ES cells were cultured in the presence of Wnt/transforming growth factor TGF‐β inhibitors and insulin‐like growth factor IGF‐1 to suppress mesoderm and reinforce presumptive ectoderm and otic lineages. The generated embryoid bodies were then differentiated in medium containing basic fibroblast growth factor (bFGF) for an additional 5 days using either suspension or adherent culture methods. Upon completion of differentiation, quantitative polymerase chain reaction analysis and immunostaining monitored the expression of otic/HC progenitor lineage markers. The results indicate that cells differentiated in suspension cultures produced cells expressing otic progenitor/HC markers at a higher efficiency compared with the production of these cell types within adherent cultures. Furthermore, we demonstrated that a fraction of these cells can incorporate into ototoxin‐injured mouse postnatal cochlea explants and express MYO7A after transplantation. Copyright © 2016 John Wiley & Sons, Ltd.     

Human embryonic stem cell-derived mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) originally isolated from marrow have multipotent differentiation potential and favorable immunomodulatory and anti-inflammatory properties that make them very attractive for regenerative cellular therapy. Cells with similar phenotypic characteristics have now been derived from almost all fetal, neonatal, and adult tissues; furthermore, more recently similar cells have also been generated from human embryonic stem cells (ESCs). Generation of MSCs from human ESCs provides an opportunity to study the developmental biology of human mesenchymal lineage generation in vitro. Generation of bone and cartilage from human ESC-derived MSCs and their functional characterization, both in vitro and in vivo, is also an active area of investigation as ESCs could provide an unlimited source of MSCs for potential repair of bone and cartilage defects. MSCs from adult sources are being investigated in numerous Phase I-III clinical trials for a wide variety of indications, mainly based on their immunomodulatory properties. Our group and others have shown MSCs derived from human ESCs possess immunomodulatory properties similar to marrow-derived MSCs. Immunomodulatory properties of ESC-derived MSCs could prove to be highly valuable for their potential clinical applications in the future as derivatives of human ESCs have already entered clinical arena in the context of Phase I clinical trials.     

ES cell therapy for Parkinson's disease

Neural transplantation, as a treatment for advanced Parkinson's disease (PD), has been studied for more than a decade due to the potential replacement of degenerated dopaminergic (DA) neurons. Several open-label studies on implantation of fetal nigral neurons revealed improvement in motor functions. However, the benefits were incomplete in double-blind trials. Progressive neural or embryonic stem (ES) cell research has raised hopes of creating novel cell replacement therapies for PD. DA neurons have been efficiently produced from primate ES cells in astrocyte-conditioned medium. Transplantation of neuronal stem cells derived from primate ES cells into a primate model of PD restored striatal DA function, suggesting ES cells are suitable donor cells.     

Three‐dimensional culture of single embryonic stem‐derived neural/stem progenitor cells in fibrin hydrogels: neuronal network formation and matrix remodelling

In an attempt to improve the efficacy of neural stem/progenitor cell (NSPC) based therapies, fibrin hydrogels are being explored to provide a favourable microenvironment for cell survival and differentiation following transplantation. In the present work, the ability of fibrin to support the survival, proliferation, and neuronal differentiation of NSPCs derived from embryonic stem (ES) cells under monolayer culture was explored. Single mouse ES‐NSPCs were cultured within fibrin (fibrinogen concentration: 6 mg/ml) under neuronal differentiation conditions up to 14 days. The ES‐NSPCs retained high cell viability and proliferated within small‐sized spheroids. Neuronal differentiation was confirmed by an increase in the levels of βIII‐tubulin and NF200 over time. At day 14, cell‐matrix constructs mainly comprised NSPCs and neurons (46.5% βIII‐tubulin⁺ cells). Gamma‐aminobutyric acid (GABA)ergic and dopaminergic/noradrenergic neurons were also observed, along with a network of synaptic proteins. The ES‐NSPCs expressed matriptase and secreted MMP‐2/9, with MMP‐2 activity increasing along time. Fibronectin, laminin and collagen type IV deposition was also detected. Fibrin gels prepared with higher fibrinogen concentrations (8/10 mg/ml) were less permissive to neurite extension and neuronal differentiation, possibly owing to their smaller pore area and higher rigidity. Overall, it is shown that ES‐NSPCs within fibrin are able to establish neuronal networks and to remodel fibrin through MMP secretion and extracellular matrix (ECM) deposition. This three‐dimensional (3D) culture system was also shown to support cell viability, neuronal differentiation and ECM deposition of human ES‐NSPCs. The settled 3D platform is expected to constitute a valuable tool to develop fibrin‐based hydrogels for ES‐NSPC delivery into the injured central nervous system. Copyright © 2016 John Wiley & Sons, Ltd.     

“The Good into the Pot, the Bad into the Crop!”—A New Technology to Free Stem Cells from Feeder Cells

A variety of embryonic and adult stem cell lines require an intial co-culturing with feeder cells for non-differentiated growth, self renewal and maintenance of pluripotency. However for many downstream ES cell applications the feeder cells have to be considered contaminations that might interfere not just with the analysis of experimental data but also with clinical application and tissue engineering approaches. Here we introduce a novel technique that allows for the selection of pure feeder-freed stem cells, following stem cell proliferation on feeder cell layers. Complete and reproducible separation of feeder and embryonic stem cells was accomplished by adaptation of an automated cell selection system that resulted in the aspiration of distinct cell colonies or fraction of colonies according to predefined physical parameters. Analyzing neuronal differentiation we demonstrated feeder-freed stem cells to exhibit differentiation potentials comparable to embryonic stem cells differentiated under standard conditions. However, embryoid body growth as well as differentiation of stem cells into cardiomyocytes was significantly enhanced in feeder-freed cells, indicating a feeder cell dependent modulation of lineage differentiation during early embryoid body development. These findings underline the necessity to separate stem and feeder cells before the initiation of in vitro differentiation. The complete separation of stem and feeder cells by this new technology results in pure stem cell populations for translational approaches. Furthermore, a more detailed analysis of the effect of feeder cells on stem cell differentiation is now possible, that might facilitate the identification and development of new optimized human or genetically modified feeder cell lines.     

Skeletal tissue engineering using embryonic stem cells

Various cell types have been investigated as candidate cell sources for cartilage and bone tissue engineering. In this review, we focused on chondrogenic and osteogenic differentiation of mouse and human embryonic stem cells (ESCs) and their potential in cartilage and bone tissue engineering. A decade ago, mouse ESCs were first used as a model to study cartilage and bone development and essential genes, factors and conditions for chondrogenesis and osteogenesis were unravelled. This knowledge, combined with data from the differentiation of adult stem cells, led to successful chondrogenic and osteogenic differentiation of mouse ESCs and later also human ESCs. Next, researchers focused on the use of ESCs for skeletal tissue engineering. Cartilage and bone tissue was formed in vivo using ESCs. However, the amount, homogeneity and stability of the cartilage and bone formed were still insufficient for clinical application. The current protocols require improvement not only in differentiation efficiency but also in ESC‐specific hurdles, such as tumourigenicity and immunorejection. In addition, some of the general tissue engineering challenges, such as cell seeding and nutrient limitation in larger constructs, will also apply for ESCs. In conclusion, there are still many challenges, but there is potential for ESCs in skeletal tissue engineering. Copyright © 2009 John Wiley & Sons, Ltd.