Search

Table S3: List of antibodies used in this study.

Supplementary Figures S1-19 and legends. Figure S1: Overexpression of HIF-1α enhances BTIC characteristics. Figure S2: Characterizations of isolated BTICs by different parameters. Figure S3: The expression of TET1 and TET3 is upregulated by hypoxia. Figure S4: HIF-1α binding to TET1 and TET3 gene promoter in HIF-1α overexpressing cells. Figure S5: Knockdown of TET diminishes hypoxia induced activation of stemness related genes in breast cancer cell lines. Figure S6: Knockdown of TET1/3 represses tumorigenicity of isolated BTICs in vivo. Figure S7: Isotype control for IHC analysis. Figure S8: Gene specific changes in 5hmC level and TET proteins binding in hypoxic, HIF-1α overexpressing cells and isolated BTICs. Figure S9: Starvation is unable to affect the levels of 5hmC on the genome regions of TNFα gene and TNFα gene expression. Figure S10: TET proteins are essential for hypoxia activated TNFα-p38-MAPK signaling. Figure S11: TET proteins are required for hypoxia-induced 5hmC change on the genome regions of the TNFα gene. Figure S12: The enzymatic activity of TET proteins is required for establishing 5hmC signature upon hypoxia. Figure S13: The enzymatic activity of TET proteins is required for activation of TNFα-p38-MAPK signaling upon hypoxia. Figure S14: Protein-protein interaction between TET1 and TET3 in hypoxic cells and isolated BTICs. Figure S15: Co-occupancy of TET1 and TET3 on genome regions of the TNFα gene. Figure S16: Suppression of p38-MAPK signaling reduces BTIC properties. Figure S17: Certain downstream targets of TNFα-p38-MAPK signaling are regulated by hypoxia/TET. Figure S18: The levels of 5hmC and the bindings of TET proteins at pluripotent loci upon hypoxia. Figure S19: The landscape for 5hmC level and TET proteins binding on pluripotent loci upon hypoxia.

Apolipoprotein E4 (APOEε4) is the major allelic risk factor for late-onset sporadic Alzheimer's disease (sAD). Inflammation is increasingly considered as critical in sAD initiation and progression. Identifying brain molecular mechanisms that could bridge these two risk factors remain unelucidated. Leveraging induced pluripotent stem cell (iPSC)-based strategies, we demonstrate that APOE controls inflammation in human astrocytes by regulating Transgelin 3 (TAGLN3) expression and, ultimately, nuclear factor κB (NF-κB) activation. We uncover that APOE4 specifically downregulates TAGLN3, involving histone deacetylases activity, which results in low-grade chronic inflammation and hyperactivated inflammatory responses. We show that APOE4 exerts a dominant negative effect to prime astrocytes toward a pro-inflammatory state that is pharmacologically reversible by TAGLN3 supplementation. We further confirm that TAGLN3 is downregulated in the brain of patients with sAD. Our findings highlight the APOE-TAGLN3-NF-κB axis regulating neuroinflammation in human astrocytes and reveal TAGLN3 as a molecular target to modulate neuroinflammation, as well as a potential biomarker for AD.

Farnesoid X receptor (FXR) agonists are emerging as important potential therapeutics for the treatment of nonalcoholic steatohepatitis (NASH) patients, as they exert positive effects on multiple aspects of the disease. FXR agonists reduce lipid accumulation in the liver, hepatocellular inflammation, hepatic injury, and fibrosis. While there are currently no approved therapies for NASH, the bile acid-derived FXR agonist obeticholic acid (OCA; 6-ethyl chenodeoxycholic acid) has shown promise in clinical studies. Previously, we described the discovery of tropifexor (LJN452), the most potent non-bile acid FXR agonist currently in clinical investigation. Here, we report the discovery of a novel chemical series of non-bile acid FXR agonists based on a tricyclic dihydrochromenopyrazole core from which emerged nidufexor (LMB763), a compound with partial FXR agonistic activity in vitro and FXR-dependent gene modulation in vivo. Nidufexor has advanced to Phase 2 human clinical trials in patients with NASH and diabetic nephropathy.

Cancer comprises heterogeneous cells, ranging from highly proliferative immature precursors to more differentiated cell lineages. The emergence of the "cancer stem cell" (CSC) hypothesis that they are the cells responsible for resistance, metastasis and secondary tumor appearance identifies these populations as novel obligatory targets for the treatment of cancer. CSCs, like their normal tissue-specific stem cell counterparts, are multipotent, partially differentiated, self-sustaining, yet transformed cells. To date, most studies on CSC biology have relied on the use of murine models and primary human material. In spite of much progress, the use of primary material presents several limitations that limit our understanding of the mechanisms underlying CSC formation, the similarities between normal stem cells and CSCs and ultimately, the possibility for developing targeted therapies. Recently, different strategies for controlling cell fate have been applied to the modeling of human cancer initiation and for the generation of human CSC models. Here we will summarize recent developments in the establishment and application of reprogramming strategies for the modeling of human cancer initiation and CSC formation.

Cancer stem cells (CSCs) have been implicated in the initiation and maintenance of tumour growth as well as metastasis. Recent reports link stemness to epithelial–mesenchymal transition (EMT) in cancer. However, there is still little knowledge about the molecular markers of those events. In silico analysis of RNA profiles of 36 pancreatic ductal adenocarcinomas (PDAC) reveals an association of the expression of CD95 with EMT and stemness that was validated in CSCs isolated from PDAC surgical specimens. CD95 expression was also higher in metastatic pancreatic cells than in primary PDAC. Pharmacological inhibition of CD95 activity reduced PDAC growth and metastasis in CSC-derived xenografts and in a murine syngeneic model. On the mechanistic level, Sck was identified as a novel molecule indispensable for CD95's induction of cell cycle progression. This study uncovers CD95 as a marker of EMT and stemness in PDAC. It also addresses the molecular mechanism by which CD95 drives tumour growth and opens tantalizing therapeutic possibilities in PDAC.

The generation of human induced pluripotent stem cells (iPS) has raised enormous expectations within the biomedical community due to their potential vast implications in regenerative and personalized medicine. However, reprogramming to iPS is still not fully comprehended. Difficulties found in ascribing specific molecular patterns to pluripotent cells (PSCs), and inherent inter-line and intra-line variability between different PSCs need to be resolved. Additionally, and despite multiple assumptions, it remains unclear whether the current in vitro culturing conditions for the maintenance and differentiation of PSCs do indeed recapitulate the developmental processes observed in vivo. As a consequence, basic questions such as what is the actual nature of PSCs remain unanswered and different theories have emerged in regards to the identity of these valuable cell population. Here we discuss on the published theories for defining PSC identity, the implications that the different postulated models have for the reprogramming field as well as speculate on potential future directions that might be opened once a precise knowledge on the nature of PSCs is accomplished.

Somatic cell nuclear reprogramming is opening new doors for the modeling of human disease phenotypes in vitro, the identification of novel therapeutic compounds and diagnostic factors as well as future autologous cell replacement therapies. Despite the potential that reprogramming technologies bring, there are remaining concerns preventing their broad application in the short-term. One of them is the safety concern associated with the use of stem cell derivatives, those generated by reprogramming or even when embryonic stem cells are employed. Here we summarize the current knowledge in the field of stem cells and reprogramming with a particular focus on the pitfalls preventing rapid translation of stem cell technologies into the clinic. We discuss the most recent findings on immunogenicity and tumorigenicity of reprogrammed cells. We additionally provide an overview on the potential applications that reprogramming approaches might bring to the immunological field and elaborate on the use of induced pluripotent stem cells (iPSCs) with pre-arranged immune receptors for the development of future immunotherapeutic approaches. The use of reprogramming approaches can represent and provide groundbreaking strategies previously unachievable for stem cell engineering aimed at modulating immune responses. In summary, we provide an overview on the different topics related to the use of stem cells and highlight the most provocative, yet perhaps currently underappreciated, aspect of combining immunological and reprogramming strategies for the treatment of human disease.

International audience; Since the initial discovery that OCT4, SOX2, KLF4, and c-MYC overexpression sufficed for the induction of pluripotency in somatic cells, methodologies replacing the original factors have enhanced our understanding of the reprogramming process. However, unlike in mouse, OCT4 has not been replaced successfully during reprogramming of human cells. Here we report on a strategy to accomplish this replacement. Through a combination of transcriptome and bioinformatic analysis we have identified factors previously characterized as being lineage specifiers that are able to replace OCT4 and SOX2 in the reprogramming of human fibroblasts. Our results show that it is possible to replace OCT4 and SOX2 simultaneously with alternative lineage specifiers in the reprogramming of human cells. At a broader level, they also support a model in which counteracting lineage specification networks underlies the induction of pluripotency.

Lineage conversion of one somatic cell type into another constitutes an attractive approach for research and clinical use. Lineage conversion can proceed in a direct manner, in the absence of proliferation and multipotent progenitor generation, or in an indirect manner, by the generation of expandable multipotent progenitor states. Here we report on the development of a combined reprogramming methodology that, transitioning through a plastic intermediate state, allows for the generation of human mesodermal progenitor cells while circumventing the traditional hallmarks of pluripotency. Converted mesodermal progenitor cells demonstrated bi-potent differentiation potential and were able to generate endothelial and smooth muscle lineages. Importantly, human fibroblasts can be converted into angioblast-like progenitor cells by non-integrative approaches. Differentiated angioblast-like cells exhibit neo-angiogenesis and anastomosis in vivo. The methodology for indirect lineage conversion to angioblast-like cells described here adds to the armamentarium of reprogramming approaches aimed at the clinical treatment of ischemic pathologies.

CpG islands (CGIs) are primarily promoter-associated genomic regions and are mostly unmethylated within highly methylated mammalian genomes. The mechanisms by which CGIs are protected from de novo methylation remain elusive. Here we show that insertion of CpG-free DNA into targeted CGIs induces de novo methylation of the entire CGI in human pluripotent stem cells (PSCs). The methylation status is stably maintained even after CpG-free DNA removal, extensive passaging, and differentiation. By targeting the DNA mismatch repair gene MLH1 CGI, we could generate a PSC model of a cancer-related epimutation. Furthermore, we successfully corrected aberrant imprinting in induced PSCs derived from an Angelman syndrome patient. Our results provide insights into how CpG-free DNA induces de novo CGI methylation and broaden the application of targeted epigenome editing for a better understanding of human development and disease.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal human premature ageing disease, characterized by premature arteriosclerosis and degeneration of vascular smooth muscle cells (SMCs). HGPS is caused by a single point mutation in the lamin A (LMNA) gene, resulting in the generation of progerin, a truncated splicing mutant of lamin A. Accumulation of progerin leads to various ageing-associated nuclear defects including disorganization of nuclear lamina and loss of heterochromatin. Here we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with HGPS. HGPS-iPSCs show absence of progerin, and more importantly, lack the nuclear envelope and epigenetic alterations normally associated with premature ageing. Upon differentiation of HGPS-iPSCs, progerin and its ageing-associated phenotypic consequences are restored. Specifically, directed differentiation of HGPS-iPSCs to SMCs leads to the appearance of premature senescence phenotypes associated with vascular ageing. Additionally, our studies identify DNA-dependent protein kinase catalytic subunit (DNAPKcs, also known as PRKDC) as a downstream target of progerin. The absence of nuclear DNAPK holoenzyme correlates with premature as well as physiological ageing. Because progerin also accumulates during physiological ageing, our results provide an in vitro iPSC-based model to study the pathogenesis of human premature and physiological vascular ageing.

SummaryInjury to the central nervous system initiates an uncontrolled inflammatory response that results in both tissue repair and destruction. Here, we showed that, in rodents and humans, injury to the spinal cord triggered surface expression of CD95 ligand (CD95L, FasL) on peripheral blood myeloid cells. CD95L stimulation of CD95 on these cells activated phosphoinositide 3-kinase (PI3K) and metalloproteinase-9 (MMP-9) via recruitment and activation of Syk kinase, ultimately leading to increased migration. Exclusive CD95L deletion in myeloid cells greatly decreased the number of neutrophils and macrophages infiltrating the injured spinal cord or the inflamed peritoneum after thioglycollate injection. Importantly, deletion of myeloid CD95L, but not of CD95 on neural cells, led to functional recovery of spinal injured animals. Our results indicate that CD95L acts on peripheral myeloid cells to induce tissue damage. Thus, neutralization of CD95L should be considered as a means to create a controlled beneficial inflammatory response.

SummaryAdult neurogenesis persists in the subventricular zone and the dentate gyrus and can be induced upon central nervous system injury. However, the final contribution of newborn neurons to neuronal networks is limited. Here we show that in neural stem cells, stimulation of the “death receptor” CD95 does not trigger apoptosis but unexpectedly leads to increased stem cell survival and neuronal specification. These effects are mediated via activation of the Src/PI3K/AKT/mTOR signaling pathway, ultimately leading to a global increase in protein translation. Induction of neurogenesis by CD95 was further confirmed in the ischemic CA1 region, in the naive dentate gyrus, and after forced expression of CD95L in the adult subventricular zone. Lack of hippocampal CD95 resulted in a reduction in neurogenesis and working memory deficits. Following global ischemia, CD95-mediated brain repair rescued behavioral impairment. Thus, we identify the CD95/CD95L system as an instructive signal for ongoing and injury-induced neurogenesis.

Glioma tumour-initiating cells (GTICs) can originate upon the transformation of neural progenitor cells (NPCs). Studies on GTICs have focused on primary tumours from which GTICs could be isolated and the use of human embryonic material. Recently, the somatic genomic landscape of human gliomas has been reported. RTK (receptor tyrosine kinase) and p53 signalling were found dysregulated in ∼90% and 86% of all primary tumours analysed, respectively. Here we report on the use of human-induced pluripotent stem cells (hiPSCs) for modelling gliomagenesis. Dysregulation of RTK and p53 signalling in hiPSC-derived NPCs (iNPCs) recapitulates GTIC properties in vitro. In vivo transplantation of transformed iNPCs leads to highly aggressive tumours containing undifferentiated stem cells and their differentiated derivatives. Metabolic modulation compromises GTIC viability. Last, screening of 101 anti-cancer compounds identifies three molecules specifically targeting transformed iNPCs and primary GTICs. Together, our results highlight the potential of hiPSCs for studying human tumourigenesis., Glioma can originate from the transformation of neural progenitor cells into glioma initiating cells. Here, the authors demonstrate the use of induced pluripotent stem cells as a suitable model for generating neural progenitor cells, which can be subsequently transformed to glioma initiating cells that are able to the generate human glioma-like tumours in mice.

Despite the profound and rapid advancements in reprogramming technologies since the generation of the first induced pluripotent stem cells (iPSCs) in 2006[1], the molecular basics of the process and its implications are still not fully understood. Recent work has suggested that a subset of TFs, so called "Pioneer TFs", play an important role during the stochastic phase of iPSC reprogramming [2-6]. Pioneer TFs activities differ from conventional transcription factors in their mechanism of action. They bind directly to condensed chromatin and elicit a series of chromatin remodeling events that lead to opening of the chromatin. Chromatin decondensation by pioneer factors progressively occurs during cell division and in turn exposes specific gene promoters in the DNA to which TFs can now directly bind to promoters that are readily accessible[2, 6]. Here, we will summarize recent advancements on our understanding of the molecular mechanisms underlying reprogramming to iPSC as well as the implications that pioneer Transcription Factor activities might play during different lineage conversion processes.

Background— Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators with important functions in development and disease. Here, we sought to identify and functionally characterize novel lncRNAs critical for vertebrate development. Methods and Results— By relying on human pluripotent stem cell differentiation models, we investigated lncRNAs differentially regulated at key steps during human cardiovascular development with a special focus on vascular endothelial cells. RNA sequencing led to the generation of large data sets that serve as a gene expression roadmap highlighting gene expression changes during human pluripotent cell differentiation. Stage-specific analyses led to the identification of 3 previously uncharacterized lncRNAs, TERMINATOR , ALIEN , and PUNISHER , specifically expressed in undifferentiated pluripotent stem cells, cardiovascular progenitors, and differentiated endothelial cells, respectively. Functional characterization, including localization studies, dynamic expression analyses, epigenetic modification monitoring, and knockdown experiments in lower vertebrates, as well as murine embryos and human cells, confirmed a critical role for each lncRNA specific for each analyzed developmental stage. Conclusions— We have identified and functionally characterized 3 novel lncRNAs involved in vertebrate and human cardiovascular development, and we provide a comprehensive transcriptomic roadmap that sheds new light on the molecular mechanisms underlying human embryonic development, mesodermal commitment, and cardiovascular specification.

Is reprogramming a physiological repair mechanism occurring during cardiac regeneration? Heart failure is one of the main health problems worldwide, and the urgent medical needs relating to it have positioned cardiovascular research as one of the most actively evolving fields in regenerative medicine. Many different strategies aiming to revert, palliate, or ameliorate the deleterious effects of heart failure have been intensively pursued, and excellent reviews on the topic exist.1–3 Briefly, approaches used to tackle cardiac repair have been focused mainly on the use of stem cells to replenish lost muscle mass (cell transplantation or mobilization of resident cardiac stem cells), reduction of cardiomyocyte hypertrophy or prevention of fibrosis, promotion of angiogenesis, and, most recently, in vivo reprogramming strategies.4–6 Although these significant advances are contributing to the development of more efficient therapies, successful and efficient heart repair strategies are by and large still lacking. Are there alternative approaches beside those mentioned above? A possible line of attack is to look at how nature deals with this issue. Regenerative animal models are able to regenerate the heart completely.7 They do so by preventing scar formation, replenishing lost muscle tissue, and eliciting a controlled proangiogenic response. All these processes lead to a complete repair of the injured heart. Whereas it is well known that these processes occur naturally only in lower vertebrates, it has been a long established dogma that the mammalian heart is not able to regenerate. However, although the adult mammalian heart is not able to regenerate after massive myocardial damage, it can activate a healing process resembling that of regenerative organisms.8 Although the numbers of newly generated cardiomyocytes are insufficient for healing of the heart, it is clear that endogenous repair mechanisms are present in the mammalian heart, thus providing a potential …

Hypoxia is a hallmark of solid tumors that drives malignant progression by altering epigenetic controls. In breast tumors, aberrant DNA methylation is a prevalent epigenetic feature associated with increased risk of metastasis and poor prognosis. However, the mechanism by which hypoxia alters DNA methylation or other epigenetic controls that promote breast malignancy remains poorly understood. We discovered that hypoxia deregulates TET1 and TET3, the enzymes that catalyze conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), thereby leading to breast tumor–initiating cell (BTIC) properties. TET1/3 and 5hmC levels were closely associated with tumor hypoxia, tumor malignancy, and poor prognosis in breast cancer patients. Mechanistic investigations showed that hypoxia leads to genome-wide changes in DNA hydroxymethylation associated with upregulation of TNFα expression and activation of its downstream p38–MAPK effector pathway. Coordinate functions of TET1 and TET3 were also required to activate TNFα–p38–MAPK signaling as a response to hypoxia. Our results reveal how signal transduction through the TET–TNFα–p38–MAPK signaling axis is required for the acquisition of BTIC characteristics and tumorigenicity in vitro and in vivo, with potential implications for how to eradicate BTIC as a therapeutic strategy. Cancer Res; 75(18); 3912–24. ©2015 AACR.

SummaryMitochondrial diseases include a group of maternally inherited genetic disorders caused by mutations in mtDNA. In most of these patients, mutated mtDNA coexists with wild-type mtDNA, a situation known as mtDNA heteroplasmy. Here, we report on a strategy toward preventing germline transmission of mitochondrial diseases by inducing mtDNA heteroplasmy shift through the selective elimination of mutated mtDNA. As a proof of concept, we took advantage of NZB/BALB heteroplasmic mice, which contain two mtDNA haplotypes, BALB and NZB, and selectively prevented their germline transmission using either mitochondria-targeted restriction endonucleases or TALENs. In addition, we successfully reduced human mutated mtDNA levels responsible for Leber’s hereditary optic neuropathy (LHOND), and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in mammalian oocytes using mitochondria-targeted TALEN (mito-TALENs). Our approaches represent a potential therapeutic avenue for preventing the transgenerational transmission of human mitochondrial diseases caused by mutations in mtDNA.PaperClip

Glioblastoma multiforme (GBM) remains the most aggressive brain tumor, fatal within two years from diagnosis in most patients. Oncolytic viruses, such as oncolytic herpes simplex viruses (oHSVs), constitute a promising therapeutic approach in cancer (1). Yet, direct delivery of oHSV has shown clinical limitations in terms of efficacy. Duebgen et al. analyzed the role of oHSVs for the treatment of GBM by employing cell delivery systems based on the use of mesenchymal stem cells (MSCs) encapsulated in synthetic extracellular matrices (sECMs). Loading of MSCs with oHSVs demonstrated statistically significant advantages over direct delivery of oHSVs, in terms of efficacy, in clinical animal models of GBM (2). Engineering of oHSVs with tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) molecules further enhanced antitumoral responses and led to increased survival. While the study by Duebgen et al. represents a substantial advancement on the development of therapies against GBM, we also find issues that require further consideration. First, MSCs have demonstrated immunomodulatory roles affecting T-cells, B-cells, and dendritic cells (DCs) (3), as well as brain immunity through regulation of microglia (4). Immune cells synthesize cytokines mediating tissue inflammation, and inflammation has been linked to immune evasion, migration, and growth of several cancer types, including brain tumors (5). Therefore, the immunodeficient xenograft models used in this study might not fully recapitulate human physiology and, accordingly, the responses that MSCs might elicit in human patients. Indeed, it is widely accepted that inflammation should be considered when aiming at developing novel anticancer therapies. As MSCs can be readily isolated from rodents, autologous immunocompetent models might provide important information parallel to the use of xenograft heterologous transplantation experiments. Therefore, evaluating the role of inflammation remains critical for the future development of autologous therapies in humans, as suggested by the authors. Second, we also wondered whether the use of oHSVs-MSCs might provide additional beneficial effects in combination with standard strategies. MSCs are sensitive to various types of anticancer drugs (6) that could provoke changes in their behavior (eg, differentiation), or even induce apoptosis. An alternative possibility is that radio and chemotherapy effectively target the differentiated tumoral mass, whereas oHSVs-MSCs might provide a beneficial effect against glioma stem cells, populations presumably resistant to conventional therapies and responsible for tumor relapse, as well as patient death upon migration into the contralateral hemisphere. Last, TNF family members have demonstrated to drive basal glioma migration in syngeneic animal models (7). However, the study by Duebgen et al. (2) relies on the use of human cancer lines that hardly recapitulate the infiltrative properties of GBM. Therefore, it would be of interest to evaluate the antitumoral responses elicited upon TRAIL-loaded oHSVs in models more faithfully recapitulating human GBM infiltrative behavior.

Reprogramming technologies have emerged as a promising approach for future regenerative medicine. Here, we report on the establishment of a novel methodology allowing for the conversion of human fibroblasts into hematopoietic progenitor-like cells with macrophage differentiation potential. SOX2 overexpression in human fibroblasts, a gene found to be upregulated during hematopoietic reconstitution in mice, induced the rapid appearance of CD34+ cells with a concomitant upregulation of mesoderm-related markers. Profiling of cord blood hematopoietic progenitor cell populations identified miR-125b as a factor facilitating commitment of SOX2-generated CD34+ cells to immature hematopoietic-like progenitor cells with grafting potential. Further differentiation toward the monocytic lineage resulted in the appearance of CD14+ cells with functional phagocytic capacity. In vivo transplantation of SOX2/miR-125b-generated CD34+ cells facilitated the maturation of the engrafted cells toward CD45+ cells and ultimately the monocytic/macrophage lineage. Altogether, our results indicate that strategies combining lineage conversion and further lineage specification by in vivo or in vitro approaches could help to circumvent long-standing obstacles for the reprogramming of human cells into hematopoietic cells with clinical potential. Stem Cells 2014;32:2923–2938

International audience; This protocol presents recently developed methodologies for the differentiation of human pluripotent stem cells (hPSCs) into ureteric bud (UB) progenitor-like cells. Differentiation of human PSCs to UB progenitor-like cells allows for the generation of chimeric kidney cultures in which the human cells can self-assemble into chimeric 3D structures in combination with embryonic mouse kidney cells over a period of 18 d. UB progenitor-like cells are generated by a two-step process that combines in vitro commitment of human PSCs, whether embryonic stem cells (ESCs) or induced PSCs (iPSCs), under chemically defined culture conditions, with ex vivo cultures for the induction of 3D organogenesis. The models described here provide new opportunities for investigating human kidney development, modeling disease, evaluating regenerative medicine strategies, as well as for toxicology studies.

Induced pluripotent stem cells (iPSCs) are created by the reprogramming of somatic cells via overexpression of certain transcription factors, such as the originally described Yamanaka factors: Oct4, Sox2, Klf4, and c-Myc (OSKM). Here we discuss recent advancements in iPSC reprogramming and introduce mathematical approaches to help map the landscape between cell states during reprogramming. Our modelization indicates that OSKM expression diminishes and/or changes potential barriers between cell states and that epigenetic remodeling facilitate these transitions. From a practical perspective, the modeling approaches outlined here allow us to predict the time necessary to create a given number of iPSC colonies or the number of reprogrammed cells generated in a given time. Additional investigations will help to further refine modeling strategies, rendering them applicable toward the study of the development and stability of cancer cells or even other reprogramming processes such as lineage conversion. Ultimately, a quantitative understanding of cell state transitions might facilitate the establishment of regenerative medicine strategies and enhance the translation of reprogramming technologies into the clinic.

c-Myc and phosphatidylinositol 3-OH kinase (PI3K) both participate in diverse cellular processes, including cell cycle control and tumorigenic transformation. They also contribute to preserving embryonic stem cell (ESC) characteristics. However, in spite of the vast knowledge, the molecular relationship between c-Myc and PI3K in ESCs is not known. Herein, we demonstrate that c-Myc and PI3K function cooperatively but independently to support ESC self-renewal when murine ESCs are cultured under conventional culture condition. Interestingly, culture of ESCs in 2i-condition including a GSK3β and MEK inhibitor renders both PI3K and Myc signaling dispensable for the maintenance of pluripotent properties. These results suggest that the requirement for an oncogenic proliferation-dependent mechanism sustained by Myc and PI3K is context dependent and that the 2i-condition liberates ESCs from the dependence of this mechanism. Stem Cells 2015;33:713–725

International audience; Heart failure is a leading cause of mortality and morbidity in the developed world, partly because mammals lack the ability to regenerate heart tissue. Whether this is due to evolutionary loss of regenerative mechanisms present in other organisms or to an inability to activate such mechanisms is currently unclear. Here we decipher mechanisms underlying heart regeneration in adult zebrafish and show that the molecular regulators of this response are conserved in mammals. We identified miR-99/100 and Let-7a/c and their protein targets smarca5 and fntb as critical regulators of cardiomyocyte dedifferentiation and heart regeneration in zebrafish. Although human and murine adult cardiomyocytes fail to elicit an endogenous regenerative response after myocardial infarction, we show that in vivo manipulation of this molecular machinery in mice results in cardiomyocyte dedifferentiation and improved heart functionality after injury. These data provide a proof of concept for identifying and activating conserved molecular programs to regenerate the damaged heart.

When the first human induced Pluripotent Stem Cells (iPSCs) were generated (Takahashi et al., 2007xTakahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. Cell. 2007; 131: 861–872Abstract | Full Text | Full Text PDF | PubMed | Scopus (7837)See all ReferencesTakahashi et al., 2007), the expectations surrounding the development of human SCNT methodologies for personalized medicine were rapidly wiped out. A recent report by Mitalipov and colleagues demonstrating the generation of human somatic cell nuclear transfer-derived embryonic stem cells (SCNT-ESCs) opens this long-standing debate once again, now with the added question of whether SCNT-ESCs will prove to be a better source than iPSCs for personalized regenerative medicine (Tachibana et al., 2013xTachibana, M., Amato, P., Sparman, M., Gutierrez, N.M., Tippner-Hedges, R., Ma, H., Kang, E., Fulati, A., Lee, H.S., Sritanaudomchai, H. et al. Cell. 2013; 153: 1228–1238Abstract | Full Text | Full Text PDF | PubMed | Scopus (259)See all ReferencesTachibana et al., 2013). To resolve this debate, researchers will need to address issues that, among others, include (1) the reproducibility, simplification, and expansion of SCNT technologies; (2) the potential of SCNT-ESCs for proper cell-type-specific differentiation and engraftment; (3) the role that maternal mitochondria might play in SCNT-ESCs; (4) the safety concerns inherently related to the use of pluripotent cells; and (5) the possibility that distinct molecular signatures underlie different pluripotent states.As is the case for iPSC derivation, somatic nuclear age, disease status, mutational load, and specific cell type may influence epigenetic plasticity and potentially compromise SCNT-ESC derivation and utilization. SCNT should thus be expanded to include comparative analyses of a variety of cell types from healthy, diseased, and aged individuals (the report by Mitalipov and colleagues focused exclusively on nuclei derived from fetal fibroblasts as well as those derived from a young patient with Leigh Syndrome). Regardless of the donor cell type, oocyte quality seems to have a determining role for successful SCNT. Molecular signatures defining oocyte quality, and accordingly their reprogramming potential, will need to be determined to enhance SCNT-ESC reproducibility, uniformity, and efficiency.It is not clear whether SCNT-ESCs will, similar to iPSCs, face issues in terms of altered differentiation potential and immature phenotypes (Robinton and Daley, 2012xRobinton, D.A. and Daley, G.Q. Nature. 2012; 481: 295–305Crossref | PubMed | Scopus (417)See all ReferencesRobinton and Daley, 2012). Abnormal migration and/or integration of differentiated cells derived from iPSCs into the target host tissue may eventually compromise functionality. These abnormalities could be due to incomplete resetting of the epigenome, acquisition of aberrant methylation patterns, and/or inefficient differentiation protocols. Since it is argued that SCNT-ESCs may display a more complete resetting of the epigenome, as SCNT reprogramming involves the establishment of embryonic imprinting patterns, it would be of enormous interest to determine whether and if so, how, SCNT-ESCs are functionally superior to iPSCs under the same differentiation conditions. Interestingly, a recent report by Hayashi and colleagues has indicated that both iPSCs and allogeneic ESCs derived from mice are capable of generating viable oocytes that result in normal offspring (Hayashi et al., 2012xHayashi, K., Ogushi, S., Kurimoto, K., Shimamoto, S., Ohta, H., and Saitou, M. Science. 2012; 338: 971–975Crossref | PubMed | Scopus (179)See all ReferencesHayashi et al., 2012). Whereas the findings by Hayashi and colleagues argue that both types of pluripotent cells present comparable differentiation potential, it is well accepted that murine and human PSCs present important differences that warrant further investigation.A potential concern related to SCNT-ESCs involves heteroplasmy, the presence of mitochondrial DNA (mtDNA) from dual origins. The mtDNA carried over during nuclear transfer may trigger immunogenicity, a controversial issue surrounding the use of iPSCs (Yamanaka, 2012xYamanaka, S. Cell Stem Cell. 2012; 10: 678–684Abstract | Full Text | Full Text PDF | PubMed | Scopus (248)See all ReferencesYamanaka, 2012). Although this issue has been debated, recent data support claims that murine SCNT-ESCs lack immunogenicity. For example, Craven and colleagues reported that donor mitochondria fall below the immune system’s detection level based on DNA amplification techniques (Craven et al., 2010xCraven, L., Tuppen, H.A., Greggains, G.D., Harbottle, S.J., Murphy, J.L., Cree, L.M., Murdoch, A.P., Chinnery, P.F., Taylor, R.W., Lightowlers, R.N. et al. Nature. 2010; 465: 82–85Crossref | PubMed | Scopus (158)See all ReferencesCraven et al., 2010). However, whether this will be the case when differentiated human SCNT-ESCs are exposed to actual immune cell recognition remains unknown. Moreover, due to the high degree of sequence variability of human mtDNA, changes may occur in the molecular interactions between nuclear and mitochondrial genomes and thus alter cellular function. If maternal mitochondria and reprogrammed somatic nuclei are able to reestablish proper interaction, SCNT-ESCs could constitute a new approach to treating mitochondrial diseases present in homoplasmy. Yet, since most mitochondrial diseases are heteroplasmic, other approaches, such as spindle transfer, isogenic correction in iPSCs, and induction of heteroplasmic shift, might be needed.Safety concerns constitute a major issue associated with the use of PSCs. With the continuous improvement of nonintegrative approaches for iPSC generation, the remaining safety issues relate to the potential transplantation of residual PSCs or the possibility of in vivo dedifferentiation. Interestingly, dedifferentiation has been shown to underlie both cell transformation and reprogramming to iPSCs, and the presence of “stem cell” signatures in cancer cells has been correlated with increased malignancy (Visvader and Lindeman, 2012xVisvader, J.E. and Lindeman, G.J. Cell Stem Cell. 2012; 10: 717–728Abstract | Full Text | Full Text PDF | PubMed | Scopus (328)See all ReferencesVisvader and Lindeman, 2012). Along this line, a major difference between both reprogramming processes is that, contrary to SCNT, iPSC generation is a long process influenced by cell proliferation. This extended timeframe opens the possibility that spontaneous mutations contributing to the dedifferentiation and/or enhanced proliferation are positively selected during reprogramming to iPSCs. If proven to be the case, selection during iPSC reprogramming would be analogous to what has been described during tumorigenesis and suggests that SCNT technologies may have the advantage of reducing the risk for oncogenic transformation. Additionally, reprogramming by SCNT or iPSC approaches will most probably generate differences at the epigenetic and/or genetic level, which may affect genetic instability and potential for malignant transformation. These questions still await experimental answers.Controversy remains as to whether iPSCs are molecularly identical to ESCs or if they represent a distinct pluripotent state (Yamanaka, 2012xYamanaka, S. Cell Stem Cell. 2012; 10: 678–684Abstract | Full Text | Full Text PDF | PubMed | Scopus (248)See all ReferencesYamanaka, 2012). To date, reprogramming has been described to occur by (1) factors present in the oocyte (SCNT), (2) factors typical of ESCs derived from the inner cells mass of the blastocyst (iPSCs), and more recently, (3) factors involved in lineage specification (iPSCs) (Shu et al., 2013xShu, J., Wu, C., Wu, Y., Li, Z., Shao, S., Zhao, W., Tang, X., Yang, H., Shen, L., Zuo, X. et al. Cell. 2013; 153: 963–975Abstract | Full Text | Full Text PDF | PubMed | Scopus (110)See all References, Montserrat et al., 2013xMontserrat, N., Nivet, E., Sancho-Martinez, I., Hishida, T., Kumar, S., Miquel, L., Cortina, C., Hishida, Y., Xia, Y., Esteban, C.R., and Izpisua Belmonte, J.C. Cell Stem Cell. 2013; 13Abstract | Full Text | Full Text PDF | PubMed | Scopus (60)See all References). This indicates that different paths to pluripotency can be used and extends the initial question about which cell source, SCNT-ESCs, iPSCs, or ESCs, would be most suitable for cell therapy. Noticeably, whereas ESCs from cloned embryos are virtually indistinguishable from those derived from fertilized ones by microarray analysis (Brambrink et al., 2006xBrambrink, T., Hochedlinger, K., Bell, G., and Jaenisch, R. Proc. Natl. Acad. Sci. USA. 2006; 103: 933–938Crossref | PubMed | Scopus (153)See all ReferencesBrambrink et al., 2006); higher resolution techniques and analysis of noncoding regions might highlight previously unexpected differences. Identification of different molecular signatures will contribute to the development of better reprogramming strategies and may lead to the elucidation of the best cell source for clinical applications.Despite some criticisms and unanswered questions on the practicality and utility of SNCT-ESCs, human SCNT technologies will probably contribute to the identification of novel reprogramming factors, which in turn may lead to more efficient generation and higher quality iPSCs. Paradoxically, identification of mechanisms facilitating iPSC reprogramming stemming from an increased understanding of human SCNT might eventually contribute to the disuse of SCNT-ESCs, which will always face ethical concerns that other reprogramming approaches avoid. Regardless of which source is deemed most useful for cell therapy, the testing and comparison of SCNT-ESCs with current PSCs will provide new vistas and opportunities for the study of the mechanisms underlying reprogramming. The hope is that these studies may eventually help the development of effective and safe stem-cell-based therapies.

International audience; Diseases affecting the kidney constitute a major health issue worldwide. Their incidence and poor prognosis affirm the urgent need for the development of new therapeutic strategies. Recently, differentiation of pluripotent cells to somatic lineages has emerged as a promising approach for disease modelling and cell transplantation. Unfortunately, differentiation of pluripotent cells into renal lineages has demonstrated limited success. Here we report on the differentiation of human pluripotent cells into ureteric-bud-committed renal progenitor-like cells. The generated cells demonstrated rapid and specific expression of renal progenitor markers on 4-day exposure to defined media conditions. Further maturation into ureteric bud structures was accomplished on establishment of a three-dimensional culture system in which differentiated human cells assembled and integrated alongside murine cells for the formation of chimeric ureteric buds. Altogether, our results provide a new platform for the study of kidney diseases and lineage commitment, and open new avenues for the future application of regenerative strategies in the clinic.

Reprogramming technologies, and particularly the generation of induced pluripotent stem cells (iPSCs), have demonstrated the possibility of personalized disease modeling in a dish. Importantly, the fact that pluripotent stem cells can give rise to all cell types of an organism, along with the technical progress allowing for their isolation, brings to mind fantasies like the fountain of youth and eternal regeneration and represents one of the most promising scientific fields with clinical implications. Furthermore, increasing evidence indicates that aging “defects” observed in patient somatic cells could be erased or alleviated by direct reprogramming towards pluripotency and rapidly recapitulated upon directed differentiation to specific cell lineages (Liu et al., Nature 472:221–225, 2011a). Thus, iPSC models of aging facilitate human aging studies by shortening the time required for physiological manifestation of aging-related defects from years, in the case of a human being, to days when stem cell models are applied. Moreover, the combination of gene-editing and iPSC models of aging will also allow for the generation of precisely targeted reporter cell lines of high value for studying normal differentiation processes and high throughput screens. However, a major concern regarding the use of iPSCs for disease modeling has to be taken into account prior to their broad application in drug discovery studies, which is that the use of patient-derived iPSCs bears another important experimental limitation, the lack of appropriate genetically matched control lines (Soldner et al., Cell 146:318–331, 2011; Liu et al., Cell Stem Cell 8:688–694, 2011b).

International audience; Reprogramming strategies allow for the generation of virtually any cell type of the human body, which could be useful for cell-based therapy. Among the different reprogramming technologies available, direct lineage conversion offers the possibility to change the phenotype of a cell type to another one without pushing cells backwards to a plastic/proliferative stage. This approach has raised the possibility to apply a similar process in vivo in order to compensate for functional cell loss. Historically, the cerebral tissue is a prime choice for developing cell-based treatments. As local pericyte accumulation is observed after central nervous system injury, it can be reasoned that this cell type might be a good candidate for the conversion into new neurons in vivo. In this article, and by focusing on recent observations from Karow and colleagues demonstrating the possibility to convert human brain-derived pericytes into functional neurons, we present a brief overview of the state of the art and attempt to offer perspective as to how these interesting laboratory findings could be translated in the clinic.

International audience; Nuclear-architecture defects have been shown to correlate with the manifestation of a number of human diseases as well as ageing. It is therefore plausible that diseases whose manifestations correlate with ageing might be connected to the appearance of nuclear aberrations over time. We decided to evaluate nuclear organization in the context of ageing-associated disorders by focusing on a leucine-rich repeat kinase 2 (LRRK2) dominant mutation (G2019S; glycine-to-serine substitution at amino acid 2019), which is associated with familial and sporadic Parkinson's disease as well as impairment of adult neurogenesis in mice. Here we report on the generation of induced pluripotent stem cells (iPSCs) derived from Parkinson's disease patients and the implications of LRRK2(G2019S) mutation in human neural-stem-cell (NSC) populations. Mutant NSCs showed increased susceptibility to proteasomal stress as well as passage-dependent deficiencies in nuclear-envelope organization, clonal expansion and neuronal differentiation. Disease phenotypes were rescued by targeted correction of the LRRK2(G2019S) mutation with its wild-type counterpart in Parkinson's disease iPSCs and were recapitulated after targeted knock-in of the LRRK2(G2019S) mutation in human embryonic stem cells. Analysis of human brain tissue showed nuclear-envelope impairment in clinically diagnosed Parkinson's disease patients. Together, our results identify the nucleus as a previously unknown cellular organelle in Parkinson's disease pathology and may help to open new avenues for Parkinson's disease diagnoses as well as for the potential development of therapeutics targeting this fundamental cell structure.

Lineage conversion has recently attracted increasing attention as a potential alternative to the directed differentiation of pluripotent cells to obtain cells of a given lineage. Different means allowing for cell identity switch have been reported. Lineage conversion relied initially on the discovery of specific transcription factors generally enriched and characteristic of the target cell, and their forced expression in cells of a different fate. This approach has been successful in various cases, from cells of the hematopoietic systems to neurons and cardiomyocytes. Furthermore, recent reports have suggested the possibility of establishing a general lineage conversion approach bypassing pluripotency. This requires a first phase of epigenetic erasure achieved by short overexpression of the factors used to reprogram cells to a pluripotent state (such as a combination of Sox2, Klf4, c-Myc and Oct4), followed by exposure to specific developmental cues. Here we present these different direct conversion methodologies and discuss their potential as alternatives to using induced pluripotent stem cells and differentiation protocols to generate cell populations of a given fate.

International audience; The tubular epithelium of the kidney is susceptible to injury from a number of different causes, including inflammatory and immune disorders, oxidative stress, and nephrotoxins, among others. Primary renal epithelial cells remain one of the few tools for studying the biochemical and physiological characteristics of the renal tubular system. Nevertheless, differentiated primary cells are not suitable for recapitulation of disease properties that might arise during embryonic kidney formation and further maturation. Thus, cellular systems resembling kidney characteristics are in urgent need to model disease as well as to establish reliable drug-testing platforms. Induced pluripotent stem cells (iPSCs) bear the capacity to differentiate into every cell lineage comprising the adult organism. Thus, iPSCs bring the possibility for recapitulating embryonic development by directed differentiation into specific lineages. iPSC differentiation ultimately allows for both disease modeling in vitro and the production of cellular products with potential for regenerative medicine. Here, we describe the rapid, reproducible, and highly efficient generation of iPSCs derived from endogenous kidney tubular renal epithelial cells with only two transcriptional factors, OCT4 and SOX2. Kidney-derived iPSCs may provide a reliable cellular platform for the development of kidney differentiation protocols allowing drug discovery studies and the study of kidney pathology.

One of the current chief goals of regenerative medicine involves finding a way to overcome the lack of efficient treatments for thousands of diseases. Recently, increasing evidence indicates that genetic defects observed in patient somatic cells could be erased and/or alleviated by direct reprogramming toward pluripotency and recapitulated upon directed differentiation to specific cell lineages. Therefore, major efforts have been put forth to establish efficient models of disease by the use of induced pluripotent stem cells (iPSCs). In addition to disease modeling, iPSC technology also paved the way for the development of methods allowing for the generation of transplantable cellular products to reduce/halt the progression of disease symptoms or even ultimately to cure diseases. It is likely that, in the near future, these two strategies will be complementary in the discovery of new alternative treatments. However and despite enormous enthusiasm, iPSC technology still presents several pitfalls that have to be circumvented. In the present work, we will summarize the present state of iPSC technologies alongside several alternatives to the use of iPSCs that could contribute to the establishment of cell therapies.

Efficient correction of hemoglobinopathy-causing mutations by homologous recombination in integration-free patient iPSCs

The formation of an adult organism could be viewed as a hierarchical process in which the initial totipotent cell, the zygote, progressively loses “potency” by differentiating into pluripotent, multipotent and unipotent states until the final terminally differentiated cells comprising tissues and organs are derived. Such a unidirectional concept trembled when four transcription factors were shown to “revert” the identity of differentiated somatic cells and reprogram them into induced pluripotent stem cells (iPSCs) 1. These findings rapidly fed clinical expectations for regenerative medicine purposes as well as for disease modeling and drug discovery studies 2. Though this discovery represented a promising breakthrough, the technologies applied raised several concerns, as thoroughly discussed in other reports 3. Importantly, and regardless of the methodology used for reprogramming, the lack of efficient differentiation protocols opened the possibility that mixed populations could interfere during in vitro studies. Furthermore, experimental assessment of pluripotency involves the formation of benign tumors, called teratomas. Thus, residual pluripotent stem cells need to be eliminated prior to transplantation in order to reduce the risk of malignant transformations 3. Up to date, efficient selection of residual pluripotent stem cells (PSCs) relied on the use of antibodies recognizing known pluripotency-related surface markers as well as tissue-specific fluorescence reporters combined with sorting procedures 4. Additionally, suicide gene strategies have also been described 5. Yet, such methods can lead to undesired cellular modifications due to antibody binding and/or alteration of the host genome.

Pluripotent stem cells possess the capacity for self-renewal and the ability to differentiate into all cell types of the three embryonic germ layers. Accordingly, given their potential use in regenerative medicine, they have long attracted the attention of researchers. Embryonic stem cells (ESCs) represent the model pluripotent stem cell population. However, the use of ESCs in regenerative therapies faces major hurdles—namely, immune rejection and the necessity to use early-stage embryos for ESC isolation. Clinical Pharmacology & Therapeutics (2011) 89 5, 746–749. doi:10.1038/clpt.2010.341

Recent development of a synthetic mRNA-based technology for efficient reprogramming to pluripotency and cell fate conversion without any modification to the genome has generated great interest among researchers and clinicians alike. It is hoped that this technology could contribute to unmet needs on several fronts of regenerative medicine, including mechanistic study of reprogramming, generation of safe induced pluripotent stem cells suitable for clinical applications, and derivation of desired cell types for cell-replacement therapy. We will discuss the technological advancements made by this synthetic mRNA methodology, its implications, as well as the challenges that lie ahead in the field of regenerative medicine.

Apoptosis or programmed cell-death is an important process involved in tissue homeostasis, development and a variety of immune responses.(1) The apoptotic program can be activated via transmembrane receptors stimulated by their cognate ligands. The presence of a well-conserved region of 80 amino acids in their intracellular tail, the Death-Domain (DD), has conferred those receptors the general name of "death receptors". Death receptors are a subfamily of the TNF receptor superfamily, which includes the TNF receptor-I (TNFR1), TRAMP, DR3/APO-3, TRAIL-receptor 1 (TRAIL-R1/DR4), TRAIL-receptor 2 (TRAIL-R1/DR5), DR6 and CD95 (Fas/Apo-1). The pro-apoptotic properties of the CD95 system have been extensively studied during the past decades. Nevertheless, CD95 has now emerged as an important activator of other major signaling pathways leading to a variety of phenotypes. In the last years, stimulation of CD95 has been described to activate the MAPK pathways p38, JNK and ERK. (2-6) CD95 has also been shown to activate the transcription factor NFkB. (67-9) However, the molecular mechanisms leading to activation of such pathways are not fully understood and their contribution to the final phenotype is still unclear. CD95 has been shown to be particularly involved in tumor cell invasion, (6) neurite sprouting and outgrowth,(5,10) as well as cell proliferation(11,12)--functions that lay to rest the general assumption of CD95 as a death receptor. In our group we have recently described a novel molecular link between CD95 and the phosphatydilinositol-3-kinase (PI3K) pathway in Glioblastoma multiforme. In the present review we will discuss the past and present knowledge of the CD95/CD95L system and its role in PI3K signaling.

SummaryInvasion of surrounding brain tissue by isolated tumor cells represents one of the main obstacles to a curative therapy of glioblastoma multiforme. Here we unravel a mechanism regulating glioma infiltration. Tumor interaction with the surrounding brain tissue induces CD95 Ligand expression. Binding of CD95 Ligand to CD95 on glioblastoma cells recruits the Src family member Yes and the p85 subunit of phosphatidylinositol 3-kinase to CD95, which signal invasion via the glycogen synthase kinase 3-β pathway and subsequent expression of matrix metalloproteinases. In a murine syngeneic model of intracranial GBM, neutralization of CD95 activity dramatically reduced the number of invading cells. Our results uncover CD95 as an activator of PI3K and, most importantly, as a crucial trigger of basal invasion of glioblastoma in vivo.

Cellular reprogramming to a stem-cell-like state is inefficient and poorly understood, despite its biomedical potential. Detailed molecular analyses of this process are now reported, and should help to overcome these limitations.

Gene-editing technologies and patient-specific induced pluripotent stem cells (iPSCs) may represent an unprecedented opportunity for merging the stem cell and traditional gene therapy fields to fulfill the promises of regenerative medicine.

Abstract 1217 The discovery of transcription factors and micro RNAs (miRNAs) capable of converting somatic cells, either into cells from different lineages or embryonic stem-like cells, has opened a road to differentiate and produce in vitro engraftable cells that could replace and fix damaged tissues. One of the most attractive and promising fields is the differentiation towards blood, considering it is a tissue without a complex tridimensional structure and that the phenotypes of the different sublineages are already well characterized. We previously reported the differentiation from induced pluripotent stem cells (iPS) towards hematopoietic cells and their derived lineages in in vitro assays. More recently, overexpression of Oct4 has been shown to allow for the transdifferentiation of human fibroblasts (HF) into blood progenitor cells albeit with compromised lymphoid differentiation capacity. However, there are no reports of successful differentiation into blood progenitors which are able to completely recover functionally immunodepleted mice. Considering the emerging role of miRNAs controlling development and differentiation of diverse cell lineages, we hypothesized that the gap missing in the current protocols to obtain hematopoietic stem cells can be filled by the data obtained by miRNA profiling of hematopoietic progenitor cells found in human Cord Blood samples (CB-HPC), which could unveil the key players in the maintenance of blood stemness. After in vitro expansion of different CB-HPC subpopulations, we have found a set of miRNAS specifically upregulated in the CD34+/CD90+ fraction, a population that has shown the highest capacity to repopulate immunodepleted mice. Upon serial testing, we found that miR-125b, previously reported to have a key role in human and murine hematopoiesis, is highly upregulated in CD34+/CD90+ CB-HPC. In particular, differentiation of iPS towards blood progenitors demonstrated that miR-125b overexpression, specifically increases the percentage and number of blood progenitors, as well as the ability to produce hematopoietic colonies after colony forming unit assay (CFU-A). In addition, following the same rationale previously reported for direct conversion of HF into blood progenitors, we overexpressed one of the transcription factors described by Yamanaka's group. The blood progenitors we obtained were deficient for the differentiation towards CD45+ cells, production of colonies in CFU-A, and showed no repopulation of immunodepleted mice. Most interestingly, the overexpression of miR-125b during the HF conversion, allowed us to obtain blood progenitors able to circumvent these deficiencies. Our preliminary results indicate that miR-125b overexpression causes a positive effect in the maintenance of blood progenitors through the downregulation of p53, a previously reported target for this miRNA in hematopoiesis. While several aspects remain to be addressed in terms of the prospective therapeutical approaches, our study proves that overexpression of miR-125b strongly enhances the differentiation and functionality of in vitro generated HPC. Disclosures: No relevant conflicts of interest to declare.

Abstract 1290 After the finding of a set of transcription factors capable of reprogram any somatic cell into an embryonic stem-like cell by Yamanaka's group a lot of effort has been put to differentiate and produce in-vitro engraftable cells that could replace and fix damaged tissues. One of the most attractive and promising fields is the differentiation towards blood, considering it is a tissue without a complex tridimensional structure and that the phenotypes of the different sublineages are already well characterized. Nonetheless, so far there are no reports of successful differentiation into blood progenitors which are able to completely recover functionally in vivo blood-depleted mice. We previously reported the differentiation from induced pluripotent stem cells (iPS) towards hematopoietic cells capable of distinguish into sub lineages in in vitro assays, while another group obtained blood precursors by transdifferentiation of fibroblasts; however a complete recovery of the hematopoietic lineages in vivo was not seen. Our hypothesis is that the gap missing in the current protocols to obtain repopulating blood stem cells can be filled by the microRNA profiling of Cord Blood (CB) progenitors, in order to find the key players in the maintenance of blood stemness. In particular, it has been shown that population with the highest capacity to be engrafted in mice is the CD34+/CD90+ from CB. Our preliminary results depict a set of miRNAs that are specifically overexpressed in the CD34+/CD90+ population from CB cells when compared against a less specific CD34+ population. These miRNAs are currently being tested as a tool to improve the efficiency of iPS differentiation and fibroblasts conversion towards blood progenitors by means of lentiviral infection of the miRNA precursors. Interestingly, we have found that these miRNAs have been previously reported to have a main role in the occurrence of Acute Myeloid Leukemia in humans and mice. These results led us to look for genes that are highly expressed in blood progenitors but also have been shown to be correlated with AML.As a safety study, we are currently evaluating the effect of overexpressing AML related factors (miRNAs and genes) when added to the established protocols to obtain blood progenitors from iPS and fibroblasts. Surprisingly, our initial results show that the overxpression of the above mentioned genes and miRNAs have an intrinsic potential to induce in vitro differentiation or conversion from iPS and fibroblasts towards blood progenitors. Disclosures: No relevant conflicts of interest to declare.

Finding a cure for cardiovascular disease remains a major unmet medical need. Recent investigations have started to unveil the mechanisms of mammalian heart regeneration. The study of the regenerative mechanisms in lower vertebrate and mammalian animal models has provided clues for the experimental activation of proregenerative responses in the heart. In parallel, the use of endogenous adult stem cell populations alongside the recent application of reprogramming technologies has created major expectations for the development of therapies targeting heart disease. Together, these new approaches are bringing us closer to more successful strategies for the treatment of heart disease.

International audience; Combination of stem cell-based approaches with gene-editing technologies represents an attractive strategy for studying human disease and developing therapies. However, gene-editing methodologies described to date for human cells suffer from technical limitations including limited target gene size, low targeting efficiency at transcriptionally inactive loci, and off-target genetic effects that could hamper broad clinical application. To address these limitations, and as a proof of principle, we focused on homologous recombination-based gene correction of multiple mutations on lamin A (LMNA), which are associated with various degenerative diseases. We show that helper-dependent adenoviral vectors (HDAdVs) provide a highly efficient and safe method for correcting mutations in large genomic regions in human induced pluripotent stem cells and can also be effective in adult human mesenchymal stem cells. This type of approach could be used to generate genotype-matched cell lines for disease modeling and drug discovery and potentially also in therapeutics.