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This year marks the tenth anniversary of the generation of induced pluripotent stem cells (iPSCs) by transcription factor-mediated somatic cell reprogramming. Takahashi and Yamanaka portray the path towards this ground-breaking discovery and discuss how, since then, research has focused on understanding the mechanisms underlying iPSC generation and on translating such advances to the clinic. The past 10 years have seen great advances in our ability to manipulate cell fate, including the induction of pluripotency in vitro to generate induced pluripotent stem cells (iPSCs). This process proved to be remarkably simple from a technical perspective, only needing the host cell and a defined cocktail of transcription factors, with four factors — octamer-binding protein 3/4 (OCT3/4), SOX2, Krüppel-like factor 4 (KLF4) and MYC (collectively referred to as OSKM) — initially used. The mechanisms underlying transcription factor-mediated reprogramming are still poorly understood; however, several mechanistic insights have recently been obtained. Recent years have also brought significant progress in increasing the efficiency of this technique, making it more amenable to applications in the fields of regenerative medicine, disease modelling and drug discovery.

CRISPR/Cas mediated genome editing has been successfully demonstrated in mammalian cells and further applications for generating mutant mice were reported by injecting humanized Cas9 ( hCas ) mRNA and single guide RNA into fertilized eggs. Here we inject the circular plasmids expressing hCas9 and sgRNA into mouse zygotes and obtained mutant mice within a month. When we targeted the Cetn1 locus, 58.8% (10/17) of the pups carried the mutations and six of them were homozygously mutated. Co-injection of the plasmids targeting different loci resulted in the successful removal of the flanked region in two out of three mutant pups. The efficient mutagenesis was also observed at the Prm1 locus. Among the 46 offspring carrying CRISPR/Cas plasmid mediated mutations, only two of them carried the hCas9 transgene. The pronuclear injection of circular plasmid expressing hCas9/sgRNA complex is a rapid, simple and reproducible method for targeted mutagenesis.

Factors limiting the adoption of iPSC technology include the cost of developing lines and the time period that it takes to characterize and bank them, particularly when integration free, feeder free and Xeno-free components are used. In this manuscript we describe our optimization procedure that enables a single technician to make 20–40 lines at a time in a 24–96 well format in a reliable and reproducible fashion. Improvements spanned the entire workflow and included using RNA virus, reducing cytotoxicity of reagents, developing improved transfection and freezing efficiencies, modifying the manual colony picking steps, enhancing passaging efficiency and developing early criteria of success. These modifications allowed us to make more than two hundred well-characterized lines per year.

The ability to control the transition from an undifferentiated stem cell to a specific cell fate is one of the key techniques that are required for the application of interventional technologies to regenerative medicine and the treatment of tumors and metastases and of neurodegenerative diseases. Reprogramming technologies, which include somatic cell nuclear transfer, induced pluripotent stem cells, and the direct reprogramming of specific cell lineages, have the potential to alter cell plasticity in translational medicine for cancer treatment. The characterization of cancer stem cells (CSCs), the identification of oncogene and tumor suppressor genes for CSCs, and the epigenetic study of CSCs and their microenvironments are important topics. This review summarizes the application of cell reprogramming technologies to cancer modeling and treatment and discusses possible obstacles, such as genetic and epigenetic alterations in cancer cells, as well as the strategies that can be used to overcome these obstacles to cancer research.

An analysis of published patent applications in chimeric antigen receptor (CAR) T-cell immunotherapy allows insight into the development of this emerging technology.

We report the differentiation of neural stem and progenitor cells solely induced by direct current (DC) pulses stimulation. Neural stem and progenitor cells in the adult mammalian brain are promising candidates for the development of therapeutic neuroregeneration strategies. The differentiation of neural stem and progenitor cells depends on various in vivo environmental factors, such as nerve growth factor and endogenous EF. In this study, we demonstrated that the morphologic and phenotypic changes of mouse neural stem and progenitor cells (mNPCs) could be induced solely by exposure to square-wave DC pulses (magnitude 300 mV/mm at frequency of 100-Hz). The DC pulse stimulation was conducted for 48 h, and the morphologic changes of mNPCs were monitored continuously. The length of primary processes and the amount of branching significantly increased after stimulation by DC pulses for 48 h. After DC pulse treatment, the mNPCs differentiated into neurons, astrocytes, and oligodendrocytes simultaneously in stem cell maintenance medium. Our results suggest that simple DC pulse treatment could control the fate of NPCs. With further studies, DC pulses may be applied to manipulate NPC differentiation and may be used for the development of therapeutic strategies that employ NPCs to treat nervous system disorders.

Advances in genetic engineering technologies have allowed the construction of artificial genetic circuits, which have been used to generate spatial patterns of differential gene expression. However, the question of how cells can be programmed, and how complex the rules need to be, to achieve a desired tissue morphology has received less attention. Here, we address these questions by developing a mathematical model to study how cells can collectively grow into clusters with different structural morphologies by secreting diffusible signals that can influence cellular growth rates. We formulate how growth regulators can be used to control the formation of cellular protrusions and how the range of achievable structures scales with the number of distinct signals. We show that a single growth inhibitor is insufficient for the formation of multiple protrusions but may be achieved with multiple growth inhibitors, and that other types of signals can regulate the shape of protrusion tips. These examples illustrate how our approach could potentially be used to guide the design of regulatory circuits for achieving a desired target structure.

Mesenchymal stem/stromal cells (MSCs) are widely studied by both academia and industry for a broad array of clinical indications. The collective body of data provides compelling evidence of the clinical safety of MSC therapy. However, generally accepted proof of therapeutic efficacy has not yet been reported. In an effort to generate a more effective therapeutic cell product, investigators are focused on modifying MSC processing protocols to enhance the intrinsic biologic activity. Here, we report a Good Manufacturing Practice‐compliant two‐step MSC manufacturing protocol to generate MSCs or interferon γ (IFNγ) primed MSCs which allows freshly expanded cells to be infused in patients on a predetermined schedule. This protocol eliminates the need to infuse cryopreserved, just thawed cells which may reduce the immune modulatory activity. Moreover, using (IFNγ) as a prototypic cytokine, we demonstrate the feasibility of priming the cells with any biologic agent. We then characterized MSCs and IFNγ primed MSCs prepared with our protocol, by karyotype, in vitro potential for malignant transformation, biodistribution, effect on engraftment of transplanted hematopoietic cells, and in vivo toxicity in immune deficient mice including a complete post‐mortem examination. We found no evidence of toxicity attributable to the MSC or IFNγ primed MSCs. Our data suggest that the clinical risk of infusing MSCs or IFNγ primed MSCs produced by our two‐step protocol is not greater than MSCs currently in practice. While actual proof of safety requires phase I clinical trials, our data support the use of either cell product in new clinical studies. Stem Cells Translational Medicine 2017;6:1868–1879 Schematic representation of a 2‐step GMP manufacturing process allowing for a reliably scheduled infusion of freshly expanded mesenchymal stem cells (MSCs).

Groundbreaking studies showed that differentiated somatic cells of mouse and human origin could be reverted to a stable pluripotent state by the ectopic expression of only four proteins. The resulting pluripotent cells, called induced pluripotent stem (iPS) cells, could be an alternative to embryonic stem cells, which are under continuous ethical debate. Hence, iPS cell-derived functional cells such as neurons may become the key for an effective treatment of currently incurable degenerative diseases. However, besides the requirement of efficacy testing of the therapy also its long-term safety needs to be carefully evaluated in settings mirroring the clinical situation in an optimal way. In this context, we chose the long-lived common marmoset monkey (Callithrix jacchus) as a non-human primate species to generate iPS cells. The marmoset monkey is frequently used in biomedical research and is gaining more and more preclinical relevance due to the increasing number of disease models. Here, we describe, to our knowledge, the first-time generation of marmoset monkey iPS cells from postnatal skin fibroblasts by non-viral means. We used the transposon-based, fully reversible piggyback system. We cloned the marmoset monkey reprogramming factors and established robust and reproducible reprogramming protocols with a six-factor-in-one-construct approach. We generated six individual iPS cell lines and characterized them in comparison with marmoset monkey embryonic stem cells. The generated iPS cells are morphologically indistinguishable from marmoset ES cells. The iPS cells are fully reprogrammed as demonstrated by differentiation assays, pluripotency marker expression and transcriptome analysis. They are stable for numerous passages (more than 80) and exhibit euploidy. In summary, we have established efficient non-viral reprogramming protocols for the derivation of stable marmoset monkey iPS cells, which can be used to develop and test cell replacement therapies in preclinical settings.

Breakthrough raises call for debate over prospect of artificial human eggs.