Induction and enhancement of cardiac cell differentiation from mouse and human induced pluripotent stem cells with cyclosporin-A

Induced pluripotent stem cells (iPSCs) are novel stem cells derived from adult mouse and human tissues by reprogramming. Elucidation of mechanisms and exploration of efficient methods for their differentiation to functional cardiomyocytes are essential for developing cardiac cell models and future regenerative therapies. We previously established a novel mouse embryonic stem cell (ESC) and iPSC differentiation system in which cardiovascular cells can be systematically induced from Flk1(+) common progenitor cells, and identified highly cardiogenic progenitors as Flk1(+)/CXCR4(+)/VE-cadherin(-) (FCV) cells. We have also reported that cyclosporin-A (CSA) drastically increases FCV progenitor and cardiomyocyte induction from mouse ESCs. Here, we combined these technologies and extended them to mouse and human iPSCs. Co-culture of purified mouse iPSC-derived Flk1(+) cells with OP9 stroma cells induced cardiomyocyte differentiation whilst addition of CSA to Flk1(+) cells dramatically increased both cardiomyocyte and FCV progenitor cell differentiation. Spontaneously beating colonies were obtained from human iPSCs by co-culture with END-2 visceral endoderm-like cells. Appearance of beating colonies from human iPSCs was increased approximately 4.3 times by addition of CSA at mesoderm stage. CSA-expanded human iPSC-derived cardiomyocytes showed various cardiac marker expressions, synchronized calcium transients, cardiomyocyte-like action potentials, pharmacological reactions, and ultra-structural features as cardiomyocytes. These results provide a technological basis to obtain functional cardiomyocytes from iPSCs.     

Qualitative and Quantitative Comparison of the Proteome of Erythroid Cells Differentiated from Human iPSCs and Adult Erythroid Cells by Multiplex TMT Labelling and NanoLC-MS/MS

Induced pluripotent stem cells (iPSC) are an attractive progenitor source for the generation of in vitro blood products. However, before iPSC-derived erythroid cells can be considered for therapeutic use their similarity to adult erythroid cells must be confirmed. We have analysed the proteome of erythroid cells differentiated from the iPSC fibroblast derived line (C19) and showed they express hallmark RBC proteins, including all those of the ankyrin and 4.1R complex. We next compared the proteome of erythroid cells differentiated from three iPSC lines (C19, OCE1, OPM2) with that of adult and cord blood progenitors. Of the 1989 proteins quantified <3% differed in level by 2-fold or more between the different iPSC-derived erythroid cells. When compared to adult cells, 11% of proteins differed in level by 2-fold or more, falling to 1.9% if a 5-fold threshold was imposed to accommodate slight inter-cell line erythropoietic developmental variation. Notably, the level of >30 hallmark erythroid proteins was consistent between the iPSC lines and adult cells. In addition, a sub-population (10–15%) of iPSC erythroid cells in each of the iPSC lines completed enucleation. Aberrant expression of some cytoskeleton proteins may contribute to the failure of the majority of the cells to enucleate since we detected some alterations in cytoskeletal protein abundance. In conclusion, the proteome of erythroid cells differentiated from iPSC lines is very similar to that of normal adult erythroid cells, but further work to improve the induction of erythroid cells in existing iPSC lines or to generate novel erythroid cell lines is required before iPSC-derived red cells can be considered suitable for transfusion therapy.     

Hepatic differentiation of mouse iPS cells and analysis of liver engraftment potential of multistage iPS progeny

Hepatocyte transplantation is considered a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs) are an unlimited source for the generation of functional hepatocytes. While several protocols that direct the differentiation of iPSCs into hepatocyte-like cells have already been reported, the liver engraftment potential of iPSC progeny obtained at each step of hepatic differentiation has not yet been thoroughly investigated. In this study, we present an efficient strategy to differentiate mouse iPSCs into hepatocyte-like cells and evaluate their liver engraftment potential at different time points of the protocol (5, 10, 15, and 20 days of differentiation). iPSCs were differentiated in the presence of cytokines, growth factors, and small molecules to finally generate hepatocyte-like cells. These iPSC-derived hepatocyte-like cells exhibited hepatocyte-associated functions, such as albumin secretion and urea synthesis. When we transplanted iPSC progeny into the spleen, we found that 15- and 20-day iPSC progeny engrafted into the livers and further acquired hepatocyte morphology. In contrast, 5- and 10-day iPSC progeny were also able to engraft but did not generate hepatocyte-like cells in vivo. Our data may aid in improving current protocols geared towards the use of iPSCs as a new source of liver-targeted cell therapies.     

Neural progenitor cells from human induced pluripotent stem cells generated less autogenous immune response

The breakthrough development of induced pluripotent stem cells(iPSCs)raises the prospect of patient-specific treatment for many diseases through the replacement of affected cells.However,whether iPSC-derived functional cell lineages generate a deleterious immune response upon auto-transplantation remains unclear.In this study,we differentiated five human iPSC lines from skin fibroblasts and urine cells into neural progenitor cells(NPCs)and analyzed their immunogenicity.Through co-culture with autogenous peripheral blood mononuclear cells(PBMCs),we showed that both somatic cells and iPSC-derived NPCs do not stimulate significant autogenous PBMC proliferation.However,a significant immune reaction was detected when these cells were co-cultured with allogenous PBMCs.Furthermore,no significant expression of perforin or granzyme B was detected following stimulation of autogenous immune effector cells(CD3+CD8 T cells,CD3+CD8+T cells or CD3 CD56+NK cells)by NPCs in both PBMC and T cell co-culture systems.These results suggest that human iPSC-derived NPCs may not initiate an immune response in autogenous transplants,and thus set a base for further preclinical evaluation of human iPSCs.     

Application of biomaterials to advance induced pluripotent stem cell research and therapy

Derived from any somatic cell type and possessing unlimited self-renewal and differentiation potential, induced pluripotent stem cells (iPSCs) are poised to revolutionize stem cell biology and regenerative medicine research, bringing unprecedented opportunities for treating debilitating human diseases. To overcome the limitations associated with safety, efficiency, and scalability of traditional iPSC derivation, expansion, and differentiation protocols, biomaterials have recently been considered. Beyond addressing these limitations, the integration of biomaterials with existing iPSC culture platforms could offer additional opportunities to better probe the biology and control the behavior of iPSCs or their progeny in vitro and in vivo. Herein, we discuss the impact of biomaterials on the iPSC field, from derivation to tissue regeneration and modeling. Although still exploratory, we envision the emerging combination of biomaterials and iPSCs will be critical in the successful application of iPSCs and their progeny for research and clinical translation.     

The tumourigenicity of iPS cells and their differentiated derivates

Induced pluripotent stem cell (iPSC) provides a promising seeding cell for regenerative medicine. However, iPSC has the potential to form teratomas after transplantation. Therefore, it is necessary to evaluate the tumorigenic risks of iPSC and all its differentiated derivates prior to use in a clinical setting. Here, murine iPSCs were transduced with dual reporter gene consisting of monomeric red fluorescent protein (mRFP) and firefly luciferase (Fluc). Undifferentiated iPSCs, iPSC derivates from induced differentiation (iPSC‐derivates), iPSC‐derivated cardiomyocyte (iPSC‐CMs) were subcutaneously injected into the back of nude mice. Non‐invasive bioluminescence imaging (BLI) was longitudinally performed at day 1, 7, 14 and 28 after transplantation to track the survival and proliferation of transplanted cells. At day 28, mice were killed and grafts were explanted to detect teratoma formation. The results demonstrated that transplanted iPSCs, iPSC‐derivates and iPSC‐CMs survived in receipts. Both iPSCs and iPSC‐derivates proliferated dramatically after transplantation, while only slight increase in BLI signals was observed in iPSC‐CM transplanted mice. At day 28, teratomas were detected in both iPSCs and iPSC‐derivates transplanted mice, but not in iPSC‐CM transplanted ones. In vitro study showed the long‐term existence of pluripotent cells during iPSC differentiation. Furthermore, when these cells were passaged in feeder layers as undifferentiated iPSCs, they would recover iPSC‐like colonies, indicating the cause for differentiated iPSC's tumourigenicity. Our study indicates that exclusion of tumorigenic cells by screening in addition to lineage‐specific differentiation is necessary prior to therapeutic use of iPSCs.     

Early dynamic changes in iPSC oxygen consumption rate predict future cardiomyocyte differentiation

Human induced pluripotent stem cells (iPSCs) hold great promise for reducing the mortality of cardiovascular disease by cellular replacement of infarcted cardiomyocytes (CMs). CM differentiation via iPSCs is a lengthy multiweek process and is highly subject to batch-to-batch variability, presenting challenges in current cell manufacturing contexts. Real-time, label-free control quality attributes (CQAs) are required to ensure efficient iPSC-derived CM manufacturing. In this work, we report that live oxygen consumption rate measurements are highly predictive CQAs of CM differentiation outcome as early as the first 72 h of the differentiation protocol with an accuracy of 93%. Oxygen probes are already incorporated in commercial bioreactors, thus methods presented in this work are easily translatable to the manufacturing setting. Detecting deviations in the CM differentiation trajectory early in the protocol will save time and money for both manufacturers and patients, bringing iPSC-derived CM one step closer to clinical use.     

Long-term survival of transplanted induced pluripotent stem cell-derived neurospheres with nerve conduit into sciatic nerve defects in immunosuppressed mice

Since the advent of induced pluripotent stem cells (iPSCs), clinical trials using iPSC-based cell transplantation therapy have been performed in various fields of regenerative medicine. We previously demonstrated that the transplantation of mouse iPSC-derived neurospheres containing neural stem/progenitor cells with bioabsorbable nerve conduits promoted nerve regeneration in the long term in murine sciatic nerve defect models. However, it remains unclear how long the grafted iPSC-derived neurospheres survived and worked after implantation. In this study, the long-term survival of the transplanted mouse iPSC-derived neurospheres with nerve conduits was evaluated in high-immunosuppressed or non-immunosuppressed mice using in vivo imaging for the development of iPSC-based cell therapy for peripheral nerve injury. Complete 5-mm long defects were created in the sciatic nerves of immunosuppressed and non-immunosuppressed mice and reconstructed using nerve conduits coated with iPSC-derived neurospheres labeled with ffLuc. The survival of mouse iPSC-derived neurospheres on nerve conduits was monitored using in vivo imaging. The transplanted iPSC-derived neurospheres with nerve conduits survived for 365 days after transplantation in the immunosuppressed allograft models, but only survived for at least 14 days in non-immunosuppressed allograft models. This is the first study to find the longest survival rate of stem cells with nerve conduits transplanted into the peripheral nerve defects using in vivo imaging and demonstrates the differences in graft survival rate between the immunosuppressed allograft model and immune responsive allograft model. In the future, if iPSC-derived neurospheres are successfully transplanted into peripheral nerve defects with nerve conduits using iPSC stock cells without eliciting an immune response, axonal regeneration will be induced due to the longstanding supportive effect of grafted cells on direct remyelination and/or secretion of trophic factors.     

A Time Course Analysis of the Electrophysiological Properties of Neurons Differentiated from Human Induced Pluripotent Stem Cells (iPSCs)

Many protocols have been designed to differentiate human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) into neurons. Despite the relevance of electrophysiological properties for proper neuronal function, little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet, understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore, we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs, from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties, including resting membrane potential, action potential, sodium and potassium channel currents, somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons, the resting membrane potential became more negative, the expression of voltage-gated sodium channels increased, the membrane became capable of generating action potentials following adequate depolarization and, at day 48–55, 50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step, of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca²⁺ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology, as electrophysiological properties of iPSC-derived neurons mature over time.     

Efficient differentiation of human iPSC‐derived mesenchymal stem cells to chondroprogenitor cells

Induced pluripotent stem cells (iPSC) hold tremendous potential for personalized cell‐based repair strategies to treat musculoskeletal disorders. To establish human iPSCs as a potential source of viable chondroprogenitors for articular cartilage repair, we assessed the in vitro chondrogenic potential of the pluripotent population versus an iPSC‐derived mesenchymal‐like progenitor population. We found the direct plating of undifferentiated iPSCs into high‐density micromass cultures in the presence of BMP‐2 promoted chondrogenic differentiation, however these conditions resulted in a mixed population of cells resembling the phenotype of articular cartilage, transient cartilage, and fibrocartilage. The progenitor cells derived from human iPSCs exhibited immunophenotypic features of mesenchymal stem cells (MSCs) and developed along multiple mesenchymal lineages, including osteoblasts, adipocytes, and chondrocytes in vitro. The data indicate the derivation of a mesenchymal stem cell population from human iPSCs is necessary to limit culture heterogeneity as well as chondrocyte maturation in the differentiated progeny. Moreover, as compared to pellet culture differentiation, BMP‐2 treatment of iPSC‐derived MSC‐like (iPSC–MSC) micromass cultures resulted in a phenotype more typical of articular chondrocytes, characterized by the enrichment of cartilage‐specific type II collagen (Col2a1), decreased expression of type I collagen (Col1a1) as well as lack of chondrocyte hypertrophy. These studies represent a first step toward identifying the most suitable iPSC progeny for developing cell‐based approaches to repair joint cartilage damage. J. Cell. Biochem. 114: 480–490, 2013. © 2012 Wiley Periodicals, Inc.     

Ten years of iPSC: clinical potential and advances in vitro hematopoietic differentiation

Ten years have passed since the first publication announcing the generation of induced pluripotent stem cells (iPSCs). Issues related to ethics, immune rejection, and cell availability seemed to be solved following this breakthrough. The development of iPSC technology allows advances in in vitro cell differentiation for cell therapy purpose and other clinical applications. This review provides a perspective on the iPSC potential for cell therapies, particularly for hematological applications. We discuss the advances in in vitro hematopoietic differentiation, the possibilities to employ iPSC in hematology studies, and their potential clinical application in hematologic diseases. The generation of red blood cells and functional T cells and the genome editing technology applied to mutation correction are also covered. We highlight some of the requirements and obstacles to be overcome before translating these cells from research to the clinic, for instance, iPSC variability, genotoxicity, the differentiation process, and engraftment. Also, we evaluate the patent landscape and compile the clinical trials in the field of pluripotent stem cells. Currently, we know much more about iPSC than in 2006, but there are still challenges that must be solved. A greater understanding of molecular mechanisms underlying the generation of hematopoietic stem cells is necessary to produce suitable and transplantable hematopoietic stem progenitor cells from iPSC.     

Induced pluripotent stem cell-derived engineered T cells,natural killer cells,macrophages, and dendritic cells in immunotherapy

T cells, natural killer (NK) cells, macrophages (Macs), and dendritic cells (DCs) are among the most common sources for immune-cell-based therapies for cancer. Antitumor activity can be enhanced in induced pluripotent stem cell (iPSC)-derived immune cells by using iPSCs as a platform for stable genetic modifications that impact immuno-activating or -suppressive signaling pathways, such as transducing a chimeric antigen receptor (CAR) or deletion of immunosuppressive checkpoint molecules. This review outlines the utility of four iPSC-derived immune-cell-based therapies, highlight the latest progress and future trends in the genome-editing strategies designed to improve efficacy, safety, and universality, and provides perspectives that compare different contexts in which each of these iPSC-derived immune cell types can be most effectively used.     

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.     

Donor cell type can influence the epigenome and differentiation potential of human induced pluripotent stem cells

We compared bona fide human induced pluripotent stem cells (iPSCs) derived from umbilical cord blood (CB) cells and neonatal keratinocytes (K). As a consequence of both incomplete erasure of tissue-specific methylation and aberrant de novo methylation, CB-iPSCs and K-iPSCs were distinct in genome-wide DNA methylation profiles and differentiation potential. Extended passage of some iPSC clones in culture did not improve their epigenetic resemblance to embryonic stem cells, implying that some human iPSCs retain a residual 'epigenetic memory' of their tissue of origin.