Methods to produce induced pluripotent stem cell-derived mesenchymal stem cells: Mesenchymal stem cells from induced pluripotent stem cells

Mesenchymal stem cells (MSCs) have received significant attention in recent years due to their large potential for cell therapy. Indeed, they secrete a wide variety of immunomodulatory factors of interest for the treatment of immune-related disorders and inflammatory diseases. MSCs can be extracted from multiple tissues of the human body. However, several factors may restrict their use for clinical applications: the requirement of invasive procedures for their isolation, their limited numbers, and their heterogeneity according to the tissue of origin or donor. In addition, MSCs often present early signs of replicative senescence limiting their expansion in vitro, and their therapeutic capacity in vivo. Due to the clinical potential of MSCs, a considerable number of methods to differentiate induced pluripotent stem cells (iPSCs) into MSCs have emerged. iPSCs represent a new reliable, unlimited source to generate MSCs (MSCs derived from iPSC, iMSCs) from homogeneous and well-characterized cell lines, which would relieve many of the above mentioned technical and biological limitations. Additionally, the use of iPSCs prevents some of the ethical concerns surrounding the use of human embryonic stem cells. In this review, we analyze the main current protocols used to differentiate human iPSCs into MSCs, which we classify into five different categories: MSC Switch, Embryoid Body Formation, Specific Differentiation, Pathway Inhibitor, and Platelet Lysate. We also evaluate common and method-specific culture components and provide a list of positive and negative markers for MSC characterization. Further guidance on material requirements to produce iMSCs with these methods and on the phenotypic features of the iMSCs obtained is added. The information may help researchers identify protocol options to design and/or refine standardized procedures for large-scale production of iMSCs fitting clinical demands.     

Generation and characterization of functional cardiomyocytes using induced pluripotent stem cells derived from human fibroblasts

We have successfully developed both spontaneous and inductive cardiomyocyte differentiation of iPS cells reprogrammed from human foreskin fibroblasts. The reprogrammed iPS cells morphologically resemble human cardiomyocytes which can beat. RT-PCR and immunostaining show that cardiac markers are expressed that are comparable to the differentiation pattern of authentic human embryonic stem cells, indicating the existence of both immature and mature differentiated cardiomyocytes. 5-Azacytidine greatly enhanced the efficiency of cardiomyocyte differentiation, whereas dimethylsulfoxide had no effect. Low serum and bone morphogenetic protein-2 marginally improved differentiation efficiency. iPS cell-derived cardiomyocytes changed their beat frequency in response to cardiac drugs, which included ion channel blockers and α/β adrenergic stimulators. Derived cardiomyocytes look promising as an in vitro system for potential drug screen and/or toxicity, making this system closer to practical use in the near future.     

Embryoid body formation from embryonic and induced pluripotent stem cells: Benefits of bioreactors

Embryonic stem (ES) cells have the ability to differentiate into all germ layers, holding great promise not only for a model of early embryonic development but also for a robust cell source for cell-replacement therapies and for drug screening. Embryoid body (EB) formation from ES cells is a common method for producing different cell lineages for further applications. However, conventional techniques such as hanging drop or static suspension culture are either inherently incapable of large scale production or exhibit limited control over cell aggregation during EB formation and subsequent EB aggregation. For standardized mass EB production, a well defined scale-up platform is necessary. Recently, novel scenario methods of EB formation in hydrodynamic conditions created by bioreactor culture systems using stirred suspension systems (spinner flasks), rotating cell culture system and rotary orbital culture have allowed large-scale EB formation. Their use allows for continuous monitoring and control of the physical and chemical environment which is difficult to achieve by traditional methods. This review summarizes the current state of production of EBs derived from pluripotent cells in various culture systems. Furthermore, an overview of high quality EB formation strategies coupled with systems for in vitro differentiation into various cell types to be applied in cell replacement therapy is provided in this review. Recently, new insights in induced pluripotent stem (iPS) cell technology showed that differentiation and lineage commitment are not irreversible processes and this has opened new avenues in stem cell research. These cells are equivalent to ES cells in terms of both self-renewal and differentiation capacity. Hence, culture systems for expansion and differentiation of iPS cells can also apply methodologies developed with ES cells, although direct evidence of their use for iPS cells is still limited.     

Methods of induced pluripotent stem cells for clinical application

Reprograming somatic cells using exogenetic gene expression represents a groundbreaking step in regenerative medicine. Induced pluripotent stem cells(i PSCs) are expected to yield novel therapies with the potential to solve many issues involving incurable diseases. In particular, applying i PSCs clinically holds the promise of addressing the problems of immune rejection and ethics that have hampered the clinical applications of embryonic stem cells. However, as i PSC research has progressed, new problems have emerged that need to be solved before the routine clinical application of i PSCs can become established. In this review, we discuss the current technologies and future problems of human i PSC generation methods for clinical use.     

Cardiac disease modeling using induced pluripotent stem cell-derived human cardiomyocytes

Causative mutations and variants associated with cardiac diseases have been found in genes encoding cardiac ion channels, accessory proteins, cytoskeletal components, junctional proteins, and signaling molecules. In most cases the functional evaluation of the genetic alterationhas been carried out by expressing the mutated proteins in in-vitro heterologous systems. While these studies have provided a wealth of functional details that have greatly enhanced the understanding of the pathological mechanisms, it has always been clear that heterologous expression of the mutant protein bears the intrinsic limitation of the lack of a proper intracellular environment and the lack of pathological remodeling. The results obtained from the application of the next generation sequencing technique to patients suffering from cardiac diseases have identified several loci, mostly in non-coding DNA regions, which still await functional analysis. The isolation and culture of human embryonic stem cells has initially provided a constant source of cells from which cardiomyocytes(CMs) can be obtained by differentiation. Furthermore, the possibility to reprogram cellular fate to a pluripotent state, has opened this process to the study of genetic diseases. Thus induced pluripotent stem cells(i PSCs) represent a completely new cellular model that overcomes the limitations of heterologous studies. Importantly, due to the possibility to keep spontaneously beating CMs in culture for several months, during which they show a certain degree of maturation/aging, this approach will also provide a system in which to address the effect of long-term expression of the mutated proteins or any other DNA mutation, in terms of electrophysiological remodeling. Moreover, since i PSC preserve the entire patients’ genetic context, the system will help the physicians in identifying the most appropriate pharmacological intervention to correct the functional alteration. This article summarizes the current knowledge of cardiac genetic diseases modelled with i PSC.     

Insight into generation of induced mesenchymal stem cells from induced pluripotent cells

Mesenchymal stem cells(MSCs)have the potential for use in cell-based regenerative therapies.Currently,hundreds of clinical trials are using MSCs for the treatment of various diseases.However,MSCs are low in number in adult tissues;they show heterogeneity depending upon the cell source and exhibit limited proliferative potential and early senescence in in vitro cultures.These factors negatively impact the regenerative potential of MSCs and therefore restrict their use for clinical applications.As a result,novel methods to generate induced MSCs(iMSCs)from induced pluripotent stem cells have been explored.The development and optimization of protocols for generation of iMSCs from induced pluripotent stem cells is necessary to evaluate their regenerative potential in vivo and in vitro.In addition,it is important to compare iMSCs with primary MSCs(isolated from adult tissues)in terms of their safety and efficacy.Careful investigation of the properties of iMSCs in vitro and their long term behavior in animals is important for their translation from bench to bedside.     

Modeling of Menkes disease via human induced pluripotent stem cells

Menkes disease (MD) is a copper-deficient neurodegenerative disorder that manifests severe neurologic symptoms such as seizures, lethargic states, and hypotonia. Menkes disease is due to a dysfunction of ATP7A, but the pathophysiology of neurologic manifestation is poorly understood during embryonic development. To understand the pathophysiology of neurologic symptoms, molecular and cellular phenotypes were investigated in Menkes disease-derived induced pluripotent stem cells (MD-iPSCs). MD-iPSCs were generated from fibroblasts of a Menkes disease patient. Abnormal reticular distribution of ATP7A was observed in MD-fibroblasts and MD-iPSCs, respectively. MD-iPSCs showed abnormal morphology in appearance during embryoid body (EB) formation as compared with wild type (WT)-iPSCs. Intriguingly, aberrant switch of E-cadherin (E-cad) to N-cadherin (N-cad) and impaired neural rosette formation were shown in MD-iPSCs during early differentiation. When extracellular copper was chelated in WT-iPSCs by treatment with bathocuprione sulfate, aberrant switch of E-cad to N-cad and impaired neuronal differentiation were observed, like in MD-iPSCs. Our results suggest that neurological defects in Menkes disease patients may be responsible for aberrant cadherin transition and impaired neuronal differentiation during early developmental stage.     

Application of mesenchymal stem cells derived from human pluripotent stem cells in regenerative medicine

Mesenchymal stem cells (MSCs) represent the most clinically used stem cells in regenerative medicine. However, due to the disadvantages with primary MSCs, such as limited cell proliferative capacity and rarity in the tissues leading to limited MSCs, gradual loss of differentiation during in vitro expansion reducing the efficacy of MSC application, and variation among donors increasing the uncertainty of MSC efficacy, the clinical application of MSCs has been greatly hampered. MSCs derived from human pluripotent stem cells (hPSC-MSCs) can circumvent these problems associated with primary MSCs. Due to the infinite self-renewal of hPSCs and their differentiation potential towards MSCs, hPSC-MSCs are emerging as an attractive alternative for regenerative medicine. This review summarizes the progress on derivation of MSCs from human pluripotent stem cells, disease modelling and drug screening using hPSC-MSCs, and various applications of hPSC-MSCs in regenerative medicine. In the end, the challenges and concerns with hPSC-MSC applications are also discussed.     

Connexin mutant embryonic stem cells and human diseases

Intercellular communication via gap junctions allows cells within multicellular organisms to share small molecules. The effect of such interactions has been elucidated using mouse gene knockout strategies. Although several mutations in human gap junction-encoding connexin (Cx) have been described, Cx mutants in mice do not always recapitulate the human disease. Among the 20 mouse Cxs, Cx26, Cx43, and Cx45 play roles in early cardiac or placental development, and disruption of the genes results in lethality that hampers further analyses. Embryonic stem cells (ESCs) that lack Cx43 or Cx45 have made analysis feasible in both in vitro differentiated cell cultures and in vivo chimeric tissues. The success of mouse ESCs studies is leading to the use of induced pluripotent stem cells to learn more about the pathogenesis of human Cx diseases. This review summarizes the current status of mouse Cx disruption models and ESC differentiation studies, and discusses their implication for understanding human Cx diseases.     

Induced pluripotent stem cells for modelling human diseases

Research into the pathophysiological mechanisms of human disease and the development of targeted therapies have been hindered by a lack of predictive disease models that can be experimentally manipulated in vitro. This review describes the current state of modelling human diseases with the use of human induced pluripotent stem (iPS) cell lines. To date, a variety of neurodegenerative diseases, haematopoietic disorders, metabolic conditions and cardiovascular pathologies have been captured in a Petri dish through reprogramming of patient cells into iPS cells followed by directed differentiation of disease-relevant cells and tissues. However, realizing the true promise of iPS cells for advancing our basic understanding of disease and ultimately providing novel cell-based therapies will require more refined protocols for generating the highly specialized cells affected by disease, coupled with strategies for drug discovery and cell transplantation.     

Microencapsulation of dopamine neurons derived from human induced pluripotent stem cells

Background

Dopamine neurons derived from induced pluripotent stem cells have been widely studied for the treatment of Parkinson's disease. However, various difficulties remain to be overcome, such as tumor formation, fragility of dopamine neurons, difficulty in handling large numbers of dopamine neurons, and immune reactions. In this study, human induced pluripotent stem cell-derived precursors of dopamine neurons were encapsulated in agarose microbeads. Dopamine neurons in microbeads could be handled without specific protocols, because the microbeads protected the fragile dopamine neurons from mechanical stress.

Methods

hiPS cells were seeded on a Matrigel-coated dish and cultured to induce differentiation into a dopamine neuronal linage. On day 18 of culture, cells were collected from the culture dishes and seeded into U-bottom 96-well plates to induce cell aggregate formation. After 5 days, cell aggregates were collected from the plates and microencapsulated in agarose microbeads. The microencapsulated aggregates were cultured for an additional 45 days to induce maturation of dopamine neurons.

Results

Approximately 60% of all cells differentiated into tyrosine hydroxylase-positive neurons in agarose microbeads. The cells released dopamine for more than 40 days. In addition, microbeads containing cells could be cryopreserved.

Conclusion

hiPS cells were successfully differentiated into dopamine neurons in agarose microbeads.

General significance

Agarose microencapsulation provides a good supporting environment for the preparation and storage of dopamine neurons.     

Insulin resistance in diabetes: The promise of using induced pluripotent stem cell technology

Insulin resistance(IR)is associated with several metabolic disorders,including type 2 diabetes(T2D).The development of IR in insulin target tissues involves genetic and acquired factors.Persons at genetic risk for T2D tend to develop IR several years before glucose intolerance.Several rodent models for both IR and T2D are being used to study the disease pathogenesis;however,these models cannot recapitulate all the aspects of this complex disorder as seen in each individual.Human pluripotent stem cells(hPSCs)can overcome the hurdles faced with the classical mouse models for studying IR.Human induced pluripotent stem cells(hiPSCs)can be generated from the somatic cells of the patients without the need to destroy a human embryo.Therefore,patient-specific hiPSCs can generate cells genetically identical to IR individuals,which can help in distinguishing between genetic and acquired defects in insulin sensitivity.Combining the technologies of genome editing and hiPSCs may provide important information about the genetic factors underlying the development of different forms of IR.Further studies are required to fill the gaps in understanding the pathogenesis of IR and diabetes.In this review,we summarize the factors involved in the development of IR in the insulin-target tissues leading to diabetes.Also,we highlight the use of hPSCs to understand the mechanisms underlying the development of IR.     

Using induced pluripotent stem cells as a tool for modelling carcinogenesis

Cancer is a highly heterogeneous group of diseases that despite improved treatments remain prevalent accounting for over 14 million new cases and 8.2 million deaths per year. Studies into the process of carcinogenesis are limited by lack of appropriate models for the development and pathogenesis of the disease based on human tissues. Primary culture of patient samples can help but is difficult to grow for a number of tissues. A potential opportunity to overcome these barriers is based on the landmark study by Yamanaka which demonstrated the ability of four factors;Oct4, Sox2, Klf4, and c-Myc to reprogram human somatic cells in to pluripotency. These cells were termed induced pluripotent stem cells(i PSCs) and display characteristic properties of embryonic stem cells. This technique has a wide range of potential uses including disease modelling, drug testing and transplantation studies. Interestingly i PSCs also share a number of characteristics with cancer cells including self-renewal and proliferation, expression of stem cell markers and altered metabolism. Recently, i PSCs have been generated from a number of human cancer cell lines and primary tumour samples from a range of cancers in an attempt to recapitulate the development of cancer and interrogate the underlying mechanisms involved. This review will outline the similarities between the reprogramming process and carcinogenesis, and how these similarities have been exploited to generate i PSC models for a number of cancers.     

T lineage differentiation from induced pluripotent stem cells

Induced pluripotent stem (iPS) cells have potential to differentiate into T lymphocytes, however, the actual ability of iPS cells to develop into T lineages is not clear. In this study, we co-cultured iPS cells on OP9 cells expressing the Notch ligand Delta-like 1 (DL1), the iPS cells differentiated into T lymphocytes. In addition, in vitro stimulation of iPS cell-derived T lymphocytes resulted in secretion of IL-2 and IFN-γ. Moreover, adoptive transfer of iPS cell-derived T lymphocytes into Rag-deficient mice reconstituted their T cell pools. These results indicate that iPS cells are able to follow the normal program of T cell differentiation.     

Genomic integrity of human induced pluripotent stem cells:Reprogramming,differentiation and applications

Ten years after the initial generation of induced pluripotent stem cells(hiPSCs)from human tissues, their potential is no longer questioned, with over 15000 publications listed on PubMed, covering various fields of research; including disease modeling, cell therapy strategies, pharmacology/toxicology screening and 3 D organoid systems. However, despite evidences that the presence of mutations in hiPSCs should be a concern, publications addressing genomic integrity of these cells represent less than 1% of the literature. After a first overview of the mutation types currently reported in hiPSCs, including karyotype abnormalities, copy number variations, single point mutation as well as uniparental disomy, this review will discuss the impact of reprogramming parameters such as starting cell type and reprogramming method on the maintenance of the cellular genomic integrity. Then, a specific focus will be placed on culture conditions and subsequent differentiation protocols and how their may also trigger genomic aberrations within the cell population of interest.Finally, in a last section, the impact of genomic alterations on the possible usages of hiPSCs and their derivatives will also be exemplified and discussed. We will also discuss which techniques or combination of techniques should be used to screen for genomic abnormalities with a particular focus on the necessary quality controls and the potential alternatives.     

Predicting differentiation potential of human pluripotent stem cells:Possibilities and challenges

The capability of human pluripotent stem cell(hPSC) lines to propagate indefinitely and differentiate into derivatives of three embryonic germ layers makes these cells be powerful tools for basic scientific research and promising agents for translational medicine. However, variations in differentiation tendency and efficiency as well as pluripotency maintenance necessitate the selection of hPSC lines for the intended applications to save time and cost. To screen the qualified cell lines and exclude problematic cell lines, their pluripotency must be confirmed initially by traditional methods such as teratoma formation or by highthroughput gene expression profiling assay. Additionally, their differentiation potential, particularly the lineage-specific differentiation propensities of hPSC lines, should be predicted in an early stage. As a complement to the teratoma assay, RNA sequencing data provide a quantitative estimate of the differentiation ability of hPSCs in vivo. Moreover, multiple scorecards have been developed based on selected gene sets for predicting the differentiation potential into three germ layers or the desired cell type many days before terminal differentiation.For clinical application of hPSCs, the malignant potential of the cells must also be evaluated. A combination of histologic examination of teratoma with quantitation of gene expression data derived from teratoma tissue provides safety-related predictive information by detecting immature teratomas, malignancy marker expression, and other parameters. Although various prediction methods are available, distinct limitations remain such as the discordance of results between different assays and requirement of a long time and high labor and cost,restricting their wide applications in routine studies. Therefore, simpler and more rapid detection assays with high specificity and sensitivity that can be used to monitor the status of hPSCs at any time and fewer targeted markers that are more specific for a given desired cell type are urgently needed.