Random Mutagenesis, Clonal Events, and Embryonic or Somatic Origin Determine the mtDNA Variant Type and Load in Human Pluripotent Stem Cells.

In this study, we deep-sequenced the mtDNA of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) and their source cells and found that the majority of variants pre-existed in the cells used to establish the lines. Early-passage hESCs carried few and low-load heteroplasmic variants, similar to those identified in oocytes and inner cell masses. The number and heteroplasmic loads of these variants increased with prolonged cell culture. The study of 120 individual cells of early- and late-passage hESCs revealed a significant diversity in mtDNA heteroplasmic variants at the single-cell level and that the variants that increase during time in culture are always passenger to the appearance of chromosomal abnormalities. We found that early-passage hiPSCs carry much higher loads of mtDNA variants than hESCs, which single-fibroblast sequencing proved pre-existed in the source cells. Finally, we show that these variants are stably transmitted during short-term differentiation.

CpG Island Methylation Correlates with the Use of Alternative Promoters for USP44 Gene Expression in Human Pluripotent Stem Cells and Testes.

Deubiquitinating enzymes may play a major regulatory role in pluripotent stem cells (PSCs), but few studies have investigated this topic. Within this family of enzymes, we found that the ubiquitin-specific peptidase USP44, is highly expressed in embryonic stem cells, induced PSCs (iPSCs), and testes as compared with differentiated progenies and somatic organs. Analysis by quantitative polymerase chain reaction and 5' RACE showed that alternate promoters are responsible for expression in PSCs and organs. We noticed seven regions of transcription initiation, some of them with cell- or tissue-specific activity. Close analysis showed that one of the promoters involved in stem cell- and testis-specific activity is differentially regulated in those tissues. At the epigenetic level, USP44 transcription was correlated with DNA methylation of a CpG island close to the main promoter region. These data imply a complex picture where regulating factors such as OCT4 may interact with other epigenetic mechanisms to regulate USP44 expression in PSCs and testes.

Translation of Expanded CGG Repeats into FMRpolyG Is Pathogenic and May Contribute to Fragile X Tremor Ataxia Syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by a limited expansion of CGG repeats in the 5' UTR of FMR1. Two mechanisms are proposed to cause FXTAS: RNA gain-of-function, where CGG RNA sequesters specific proteins, and translation of CGG repeats into a polyglycine-containing protein, FMRpolyG. Here we developed transgenic mice expressing CGG repeat RNA with or without FMRpolyG. Expression of FMRpolyG is pathogenic, while the sole expression of CGG RNA is not. FMRpolyG interacts with the nuclear lamina protein LAP2ß and disorganizes the nuclear lamina architecture in neurons differentiated from FXTAS iPS cells. Finally, expression of LAP2ß rescues neuronal death induced by FMRpolyG. Overall, these results suggest that translation of expanded CGG repeats into FMRpolyG alters nuclear lamina architecture and drives pathogenesis in FXTAS.

What is really driving cell-surface interactions? Layer-by-layer assembled films may help to answer questions concerning cell attachment and response to biomaterials.

Layer-by-layer (LbL) assembled multicomponent films offer the opportunity to control and to fine-tune cell attachment and behavior on solid surfaces [Layer-by-Layer Films for Biomedical Applications, edited by Picart et al. (Wiley, Weinheim, 2014) and El-Khouri et al., "Multifunctional layer-by-layer architectures for biological applications," in Functional Polymeric Ultrathin Films, edited by Advincula and Knoll (Wiley, Weinheim, 2011), Vol. 1]. At the same time, these films allow for quite detailed physicochemical characterization of static and dynamic surface properties that are typically not available in classic cell culture. In this report, the authors investigate cell adhesion and cytocompatibility of compositionally and morphologically similar thin films composed of oppositely charged synthetic or natural polyelectrolytes in which different physical parameters such as surface charge or water content are varied through chemical composition and deposition conditions. Human adult dermal fibroblasts were chosen as a model because of the need for chemically defined matrix in the field of primary cell amplification. The growth and the stability of the multilayer films in the incubation media were studied dissipation-enhanced quartz crystal micobalance (QCM-D) and ellipsometry. The QCM-D signals observed during the film deposition were analyzed qualitatively to estimate the viscoelastic properties of the films. The authors used contact angle measurements with water to study the contribution of the chemical functionalities to wetting behavior of the films. Most importantly, they also studied the interaction of the films with serum components. Our results underline that cell adhesion is a highly complex process which is not only governed by the functionality of a surface but also by its morphology, its affinity for serum components, and also by changes of surface properties brought about by adsorbing molecules. Of the many LbL-films tested, poly(4-styrenesulfonate)/poly(allyl amine) multilayers were best suited for our fibroblast cultures, which opens a way to avoid gelatin based and similar substrates whose exact chemical composition is unknown.

Efficient and cost-effective generation of mature neurons from human induced pluripotent stem cells.

For years, our ability to study pathological changes in neurological diseases has been hampered by the lack of relevant models until the recent groundbreaking work from Yamanaka's group showing that it is feasible to generate induced pluripotent stem cells (iPSCs) from human somatic cells and to redirect the fate of these iPSCs into differentiated cells. In particular, much interest has focused on the ability to differentiate human iPSCs into neuronal progenitors and functional neurons for relevance to a large number of pathologies including mental retardation and behavioral or degenerative syndromes. Current differentiation protocols are time-consuming and generate limited amounts of cells, hindering use on a large scale. We describe a feeder-free method relying on the use of a chemically defined medium that overcomes the need for embryoid body formation and neuronal rosette isolation for neuronal precursors and terminally differentiated neuron production. Four days after induction, expression of markers of the neurectoderm lineage is detectable. Between 4 and 7 days, neuronal precursors can be expanded, frozen, and thawed without loss of proliferation and differentiation capacities or further differentiated. Terminal differentiation into the different subtypes of mature neurons found in the human brain were observed. At 6-35 days after induction, cells express typical voltage-gated and ionotrophic receptors for GABA, glycine, and acetylcholine. This specific and efficient single-step strategy in a chemically defined medium allows the production of mature neurons in 20-40 days with multiple applications, especially for modeling human pathologies.

ONSL and OSKM cocktails act synergistically in reprogramming human somatic cells into induced pluripotent stem cells.

The advent of human induced pluripotent stem cells (hiPSC) is revolutionizing many research fields including cell-replacement therapy, drug screening, physiopathology of specific diseases and more basic research such as embryonic development or diseases modeling. Despite the large number of reports on reprogramming methods, techniques in use remain globally inefficient. We present here a new optimized approach to improve this efficiency. After having tested different monocistronic vectors with poor results, we adopted a polycistronic cassette encoding Thomson's cocktail OCT4, NANOG, SOX2 and LIN28 (ONSL) separated by 2A peptides. This cassette was tested in various vector backbones, based on lentivirus or retrovirus under a LTR or EF1 alpha promoter. This allowed us to show that ONSL-carrier retrovectors reprogrammed adult fibroblast cells with a much higher efficiency (up to 0.6%) than any other tested. We then compared the reprogramming efficiencies of two different polycistronic genes, ONSL and OCT4, SOX2, KLF4 and cMYC (OSKM) placed in the same retrovector backbone. Interestingly, in this context ONSL gene reprograms more efficiently than OSKM but OSKM reprograms faster suggesting that the two cocktails may reprogram through distinct pathways. By equally mixing RV-LTR-ONSL and RV-LTR-OSKM, we indeed observed a remarkable synergy, yielding a reprogramming efficiency of >2%. We present here a drastic improvement of the reprogramming efficiency, which opens doors to the development of automated and high throughput strategies of hiPSC production. Furthermore, non-integrative reprogramming protocols (i.e. mRNA) may take advantage of this synergy to boost their efficiency.

The mammalian-specific Tex19.1 gene plays an essential role in spermatogenesis and placenta-supported development.

STUDY QUESTION: What is the consequence of Tex19.1 gene deletion in mice? SUMMARY ANSWER: The Tex19.1 gene is important in spermatogenesis and placenta-supported development. WHAT IS KNOWN ALREADY: Tex19.1 is expressed in embryonic stem (ES) cells, primordial germ cells (PGCs), placenta and adult gonads. Its invalidation in mice leads to a variable impairment in spermatogenesis and reduction of perinatal survival. STUDY DESIGN, SIZE, DURATION: We generated knock-out mice and ES cells and compared them with wild-type counterparts. The phenotype of the Tex19.1 knock-out mouse line was investigated during embryogenesis, fetal development and placentation as well as during adulthood. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used a mouse model system to generate a mutant mouse line in which the Tex19.1 gene was deleted in the germline. We performed an extensive analysis of Tex19.1-deficient ES cells and assessed their in vivo differentiation potential by generating chimeric mice after injection of the ES cells into wild-type blastocysts. For mutant animals, a morphological characterization was performed for testes and ovaries and placenta. Finally, we characterized semen parameters of mutant animals and performed real-time RT-PCR for expression levels of retrotransposons in mutant testes and ES cells. MAIN RESULTS AND THE ROLE OF CHANCE: While Tex19.1 is not essential in ES cells, our study points out that it is important for spermatogenesis and for placenta-supported development. Furthermore, we observed an overexpression of the class II LTR-retrotransposon MMERVK10C in Tex19.1-deficient ES cells and testes. LIMITATIONS, REASONS FOR CAUTION: The Tex19.1 knock-out phenotype is variable with testis morphology ranging from severely altered (in sterile males) to almost indistinguishable compared with the control counterparts (in fertile males). This variability in the testis phenotype subsequently hampered the molecular analysis of mutant testes. Furthermore, these results were obtained in the mouse, which has a second isoform (i.e. Tex19.2), while other mammals possess only one Tex19 (e.g. in humans). WIDER IMPLICATIONS OF THE FINDINGS: The fact that one gene has a role in both placentation and spermatogenesis might open new ways of studying human pathologies that might link male fertility impairment and placenta-related pregnancy disorders. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM) (Grant Avenir), the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche, the Université de Strasbourg, the Association Française contre les Myopathies (AFM) and the Fondation pour la Recherche Médicale (FRM) and Hôpitaux Universitaires de Strasbourg.The authors have nothing to disclose.

Neurons and cardiomyocytes derived from induced pluripotent stem cells as a model for mitochondrial defects in Friedreich's ataxia.

Friedreich's ataxia (FRDA) is a recessive neurodegenerative disorder commonly associated with hypertrophic cardiomyopathy. FRDA is due to expanded GAA repeats within the first intron of the gene encoding frataxin, a conserved mitochondrial protein involved in iron-sulphur cluster biosynthesis. This mutation leads to partial gene silencing and substantial reduction of the frataxin level. To overcome limitations of current cellular models of FRDA, we derived induced pluripotent stem cells (iPSCs) from two FRDA patients and successfully differentiated them into neurons and cardiomyocytes, two affected cell types in FRDA. All FRDA iPSC lines displayed expanded GAA alleles prone to high instability and decreased levels of frataxin, but no biochemical phenotype was observed. Interestingly, both FRDA iPSC-derived neurons and cardiomyocytes exhibited signs of impaired mitochondrial function, with decreased mitochondrial membrane potential and progressive mitochondrial degeneration, respectively. Our data show for the first time that FRDA iPSCs and their neuronal and cardiac derivatives represent promising models for the study of mitochondrial damage and GAA expansion instability in FRDA.

Human induced pluripotent stem cells improve stroke outcome and reduce secondary degeneration in the recipient brain.

Human induced pluripotent stem cells (hiPSCs) are a most appealing source for cell replacement therapy in acute brain lesions. We evaluated the potential of hiPSC therapy in stroke by transplanting hiPSC-derived neural progenitor cells (NPCs) into the postischemic striatum. Grafts received host tyrosine hydroxylase-positive afferents and contained developing interneurons and homotopic GABAergic medium spiny neurons that, with time, sent axons to the host substantia nigra. Grafting reversed stroke-induced somatosensory and motor deficits. Grafting also protected the host substantia nigra from the atrophy that follows disruption of reciprocal striatonigral connections. Graft innervation by tyrosine hydoxylase fibers, substantia nigra protection, and somatosensory functional recovery were early events, temporally dissociated from the slow maturation of GABAergic neurons in the grafts and innervation of substantia nigra. This suggests that grafted hiPSC-NPCs initially exert trophic effects on host brain structures, which precede integration and potential pathway reconstruction. We believe that transplantation of NPCs derived from hiPSCs can provide useful interventions to limit the functional consequences of stroke through both neuroprotective effects and reconstruction of impaired pathways.

A dual role of linker histone H1.4 Lys 34 acetylation in transcriptional activation.

The linker histone H1 is a key player in chromatin organization, yet our understanding of the regulation of H1 functions by post-translational modifications is very limited. We provide here the first functional characterization of H1 acetylation. We show that H1.4K34 acetylation (H1.4K34ac) is mediated by GCN5 and is preferentially enriched at promoters of active genes, where it stimulates transcription by increasing H1 mobility and recruiting a general transcription factor. H1.4K34ac is dynamic during spermatogenesis and marks undifferentiated cells such as induced pluripotent stem (iPS) cells and testicular germ cell tumors. We propose a model for H1.4K34ac as a novel regulator of chromatin function with a dual role in transcriptional activation.

Extended passaging increases the efficiency of neural differentiation from induced pluripotent stem cells.

BACKGROUND: The use of induced pluripotent stem cells (iPSCs) for the functional replacement of damaged neurons and in vitro disease modeling is of great clinical relevance. Unfortunately, the capacity of iPSC lines to differentiate into neurons is highly variable, prompting the need for a reliable means of assessing the differentiation capacity of newly derived iPSC cell lines. Extended passaging is emerging as a method of ensuring faithful reprogramming. We adapted an established and efficient embryonic stem cell (ESC) neural induction protocol to test whether iPSCs (1) have the competence to give rise to functional neurons with similar efficiency as ESCs and (2) whether the extent of neural differentiation could be altered or enhanced by increased passaging. RESULTS: Our gene expression and morphological analyses revealed that neural conversion was temporally delayed in iPSC lines and some iPSC lines did not properly form embryoid bodies during the first stage of differentiation. Notably, these deficits were corrected by continual passaging in an iPSC clone. iPSCs with greater than 20 passages (late-passage iPSCs) expressed higher expression levels of pluripotency markers and formed larger embryoid bodies than iPSCs with fewer than 10 passages (early-passage iPSCs). Moreover, late-passage iPSCs started to express neural marker genes sooner than early-passage iPSCs after the initiation of neural induction. Furthermore, late-passage iPSC-derived neurons exhibited notably greater excitability and larger voltage-gated currents than early-passage iPSC-derived neurons, although these cells were morphologically indistinguishable. CONCLUSIONS: These findings strongly suggest that the efficiency neuronal conversion depends on the complete reprogramming of iPSCs via extensive passaging.

Red blood cell generation from human induced pluripotent stem cells: perspectives for transfusion medicine.

BACKGROUND: Ex vivo manufacture of red blood cells from stem cells is a potential means to ensure an adequate and safe supply of blood cell products. Advances in somatic cell reprogramming of human induced pluripotent stem cells have opened the door to generating specific cells for cell therapy. Human induced pluripotent stem cells represent a potentially unlimited source of stem cells for erythroid generation for transfusion medicine. DESIGN AND METHODS: We characterized the erythroid differentiation and maturation of human induced pluripotent stem cell lines obtained from human fetal (IMR90) and adult fibroblasts (FD-136) compared to those of a human embryonic stem cell line (H1). Our protocol comprises two steps: (i) differentiation of human induced pluripotent stem cells by formation of embryoid bodies with indispensable conditioning in the presence of cytokines and human plasma to obtain early erythroid commitment, and (ii) differentiation/maturation to the stage of cultured red blood cells in the presence of cytokines. The protocol dispenses with major constraints such as an obligatory passage through a hematopoietic progenitor, co-culture on a cellular stroma and use of proteins of animal origin. RESULTS: We report for the first time the complete differentiation of human induced pluripotent stem cells into definitive erythrocytes capable of maturation up to enucleated red blood cells containing fetal hemoglobin in a functional tetrameric form. CONCLUSIONS: Red blood cells generated from human induced pluripotent stem cells pave the way for future development of allogeneic transfusion products. This could be done by banking a very limited number of red cell phenotype combinations enabling the safe transfusion of a great number of immunized patients.

High-efficiency derivation of human embryonic stem cell lines following pre-implantation genetic diagnosis.

Pre-implantation genetic diagnosis allows the characterisation of embryos that carry a gene responsible for a severe monogenic disease and to transfer to the mother's uterus only the unaffected one(s). The genetically affected embryos can be used to establish human embryonic stem cell (hESC) lines. We are currently establishing a cell bank of ESC lines carrying specific disease-causing mutant genes. These cell lines are available to the scientific community. For this purpose, we have designed a technique that requires only minimal manipulation of the embryos. At the blastocyst stage, we just removed the zona pellucida before seeding the embryo as a whole on a layer of feeder cells. This approach gave a good success rate (>20%), whatever the quality of the embryos, and allowed us to derive 11 new hESC lines, representing seven different pathologies. Full phenotypic validation of the cell lines according to ISCI guidelines confirmed their pluripotent nature, as they were positive for hESC markers and able to differentiate in vitro in all three germ layers derivatives. Nine out of 11 stem cell lines had normal karyotypes. Our results indicate that inner cell mass isolation is not mandatory for hESC derivation and that minimal manipulation of embryos can lead to high success rate.

The pluripotency-associated gene Dppa4 is dispensable for embryonic stem cell identity and germ cell development but essential for embryogenesis.

Dppa4 (developmental pluripotency-associated 4) has been identified in several high-profile screens as a gene that is expressed exclusively in pluripotent cells. It encodes a nuclear protein with an SAP-like domain and appears to be associated preferentially with transcriptionally active chromatin. Its exquisite expression pattern and results of RNA interference experiments have led to speculation that Dppa4, as well as its nearby homolog Dppa2, might play essential roles in embryonic stem (ES) cell function and/or germ cell development. To rigorously assess suggested roles, we have generated Dppa4-deficient and Dppa4/Dppa2 doubly deficient ES cells, as well as mice lacking Dppa4. Contrary to predictions, we find that Dppa4 is completely dispensable for ES cell identity and germ cell development. Instead, loss of Dppa4 in mice results in late embryonic/perinatal death and striking skeletal defects with partial penetrance. Thus, surprisingly, Dppa4-deficiency affects tissues that apparently never transcribed the gene, and at least some loss-of-function defects manifest phenotypically at an embryonic stage long after physiologic Dppa4 expression has ceased. Concomitant with targeted gene inactivation, we have introduced into the Dppa4 locus a red fluorescent marker (tandem-dimer red fluorescent protein) that is compatible with green fluorescent proteins and allows noninvasive visualization of pluripotent cells and reprogramming events.

Functional neuronal differentiation of bone marrow-derived mesenchymal stem cells.

Recent results have shown the ability of bone marrow cells to migrate in the brain and to acquire neuronal or glial characteristics. In vitro, bone marrow-derived MSCs can be induced by chemical compounds to express markers of these lineages. In an effort to set up a mouse model of such differentiation, we addressed the neuronal potentiality of mouse MSCs (mMSCs) that we recently purified. These cells expressed nestin, a specific marker of neural progenitors. Under differentiating conditions, mMSCs display a distinct neuronal shape and express neuronal markers NF-L (neurofilament-light, or neurofilament 70 kDa) and class III beta-tubulin. Moreover, differentiated mMSCs acquire neuron-like functions characterized by a cytosolic calcium rise in response to various specific neuronal activators. Finally, we further demonstrated for the first time that clonal mMSCs and their progeny are competent to differentiate along the neuronal pathway, demonstrating that these bone marrow-derived stem cells share characteristics of widely multipotent stem cells unrestricted to mesenchymal differentiation pathways.

Development of gliomas: potential role of asymmetrical cell division of neural stem cells.

Asymmetrical cell division is a mechanism that gives rise to two daughter cells with different proliferative and differentiative fates. It occurs mainly during development and in adult stem cells. Accumulating evidence suggests that tumour cells arise from the transformation of normal stem cells. Here, we propose that the asymmetrical mitosis potential of stem cells is associated with the generation of migrating tumour progenitors. Application of this speculative model to glioma proposes that the sites where tumour-initiating stem cells reside are indolent and distinct from the tumour mass, and implies that the tumour mass is continuously replenished with new migrating tumour cells from these clinically silent regions. This hypothesis offers explanations for our inability to cure glioblastoma and points to asymmetrical division as a new potential therapeutic target.

Isolation and characterisation of mesenchymal stem cells from adult mouse bone marrow.

The future use of adult mesenchymal stem cells (MSCs) for human therapies depends on the establishment of preclinical studies with other mammals such as mouse. Surprisingly, purification and characterisation of murine MSCs were only poorly documented. The aim of this study was to purify mouse MSCs from adult bone marrow and to functionally characterise their abilities to differentiate along diverse lineages. Adherent cells from adult C57Bl/6J mouse bone marrow were depleted of granulo-monocytic cells and subsequently allowed to grow on fibronectin-coated dishes in presence of fetal bovine serum and growth factors. The growing fibroblastoid cell population primarily consisted of spindle- and star-shaped cells with significant renewal capacity as they were cultured until 30 passages (about 60 doubling population). We fully demonstrated the MSC phenotype of these cells by inducing them to differentiate along osteoblastic, adipocytic, and chondrocytic pathways. Mouse MSCs (mMSCs) sharing the same morphological and functional characteristics as human MSCs can be successfully isolated from adult bone marrow without previous mouse or bone marrow treatment. Therefore, mMSCs will be an important tool to study the in vivo behaviour and fate of this cell type after grafting in mouse pathology models.

Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals.

Mesenchymal stem cells (MSCs) are largely studied for their potential clinical use. Recently, they have gained further interest after demonstration of an immunosuppressive role. In this study, we investigated whether in vivo injection of MSCs could display side effects related to systemic immunosuppression favoring tumor growth. We first showed in vitro that the murine C3H10T1/2 (C3) MSC line and primary MSCs exhibit immunosuppressive properties in mixed lymphocyte reaction. We demonstrated that this effect is mediated by soluble factors, secreted only on "activation" of MSCs in the presence of splenocytes. Moreover, the immunosuppression is mediated by CD8+ regulatory cells responsible for the inhibition of allogeneic lymphocyte proliferation. We then demonstrated that the C3 MSCs expressing the human bone morphogenetic protein 2 (hBMP-2) differentiation factor were not rejected when implanted in various allogeneic immunocompetent mice and were still able to differentiate into bone. Importantly, using a murine melanoma tumor model, we showed that the subcutaneous injection of B16 melanoma cells led to tumor growth in allogeneic recipients only when MSCs were coinjected. Although the potential side effects of immunosuppression induced by MSCs have to be considered in further clinical studies, the usefulness of MSCs for various therapeutic applications still remains of great interest.