Pluripotent Stage Research Articles

Analysis of a Minimal Gene Regulatory Network for Cell Differentiation

In this letter, we provide a detailed analysis of a gene regulatory network exhibiting bistability within a certain region of parameter space. This network has been adopted in recent literature to describe cellular differentiation into two subpopulations. Biological and experimental evidence suggests that differentiation from stem cells into different tissues evolves through a cascade of similar stages characterized by differentiation into two subtypes. Each differentiation step is influenced by two mechanisms: the first one occurs within the cell and allows transition from an undifferentiated stage to a pluripotent stage where differentiation is possible and the second mechanism may be external to the cell and biases differentiation into a specific subpopulation. In this letter, we address the former mechanism for a gene circuit described by generalized Hill equations and endowed with mutual inhibitory feedback among two competing genes. The main contribution is twofold: 1) mutual inhibition is not sufficient to allow cell differentiation, but specific conditions on the generalized Hill equations’ parameters are required and 2) differentiation occurs only if the triggering gene expression level belongs to a well defined range. Theoretical analysis is complemented with numerical simulations.

Potential Strategies for Cardiac Diseases: Lineage Reprogramming of Somatic Cells into Induced Cardiomyocytes.

Lineage reprogramming has become a potential strategy for therapy of cardiac diseases. Somatic cells can be directly converted into the induced cardiomyocytes (iCMs) without passing through an induced pluripotent stem cell stage; this strategy has some advantages such as directional differentiation and preferable security. However, there are still many challenges which need to be further studied, such as identification of safer induced factors, exploration of molecular mechanisms, improvement of the mature level of iCMs and so on. Therefore, the structures of key factors, including transcription factors, microRNAs (miRNAs), epigenetic regulators and small molecules and their functions in the cardiac development and lineage reprogramming, molecular mechanisms underlying lineage conversion, strategies for generating matured iCMs, and major challenges were reviewed to lay the foundation for further applications of iCMs.

SMCHD1 is involved in de novo methylation of the DUX4-encoding D4Z4 macrosatellite.

The DNA methylation epigenetic signature is a key determinant during development. Rules governing its establishment and maintenance remain elusive especially at repetitive sequences, which account for the majority of methylated CGs. DNA methylation is altered in a number of diseases including those linked to mutations in factors that modify chromatin. Among them, SMCHD1 (Structural Maintenance of Chromosomes Hinge Domain Containing 1) has been of major interest following identification of germline mutations in Facio-Scapulo-Humeral Dystrophy (FSHD) and in an unrelated developmental disorder, Bosma Arhinia Microphthalmia Syndrome (BAMS). By investigating why germline SMCHD1 mutations lead to these two different diseases, we uncovered a role for this factor in de novo methylation at the pluripotent stage. SMCHD1 is required for the dynamic methylation of the D4Z4 macrosatellite upon reprogramming but seems dispensable for methylation maintenance. We find that FSHD and BAMS patient's cells carrying SMCHD1 mutations are both permissive for DUX4 expression, a transcription factor whose regulation has been proposed as the main trigger for FSHD. These findings open new questions as to what is the true aetiology for FSHD, the epigenetic events associated with the disease thus calling the current model into question and opening new perspectives for understanding repetitive DNA sequences regulation.

Modeling FXS: Human Pluripotent Stem Cells and In Vitro Neural Differentiation.

In fragile X syndrome (FXS) embryos FMRP is widely expressed during early stages of embryogenesis however it is inactivated by the end of the first trimester. In the same manner, human embryonic stem cell (hESC) lines from FXS blastocysts, bearing the full CGG expansion mutation, express FMRP in their pluripotent stage and in neurons derived following in vitro differentiation, FMR1 is completely silenced. Therefore, in vitro neural differentiation of FX-hESC lines serves as a uniquely valuable model system to study the developmental mechanisms underlying FXS, together with the proper differentiation protocol to mimic the neurodevelopmental process occurs in vivo.

Modeling rare pediatric neurogenetic disorders with IPSCs

Intensive research has been performed to identify the pathological mechanisms of many pediatric neurogenetic disorders and to identify potential therapeutic targets. Although research into many pediatric neurological disorders has provided tremendous insight into the mechanisms of disease, effective treatments remain elusive. A significant impediment to progress has been a lack of thorough disease models. Transgenic/knockout animal models have been very valuable in determining the mechanisms of many neurogenetic disorders; however, these models cannot always mimic human-specific pathology and can be inadequate in representing human pathogenesis. This can be especially true for diseases of the nervous system. Alternatively, human patient-derived nervous tissue can be dangerous to acquire and difficult to propagate. The development of patient-derived induced pluripotent stem cells (IPSCs) has given researchers a fresh means of modeling these disorders with renewable human cells that can be used to generate neurons and glia. IPSCs are somatic cells that are reprogrammed back to a pluripotent stage, which can provide an unlimited source of human cells possessing patient-specific genetic mutations. Their potential to be differentiated into any cell type enables them to be a flexible platform to investigate neurogenetic disease. Of course, efficient methods for differentiating IPSCs into homogeneous populations of somatic cells must be established to provide the “disease-in-a-dish” systems. We will discuss the current methods for generating IPSC-derived neural cells to model pediatric neurogenetic disorders, as well as provide examples of the disorders that have been studied that include several neurodevelopmental and neurodegenerative disorders (Rett syndrome, spinal muscular atrophy, hereditary spastic paraplegias, and leukodystrophies). In addition, we provide examples on how patient-specific neural cells can be used in therapeutic development with high-throughput drug screening platforms or with correction via genome editing.

Reprogramming of Human Melanocytes and Melanoma Cells with Yamanaka Factors.

The expression of Yamanaka factors (Oct3/4, Klf-4, Sox-2, c-Myc) can reprogram cancer cells to a pluripotent stage. This may cause the removal of their epigenetic memory and result in altered tumorigenicity. Various studies in the literature have shown that cancer cell reprogramming is a potential tool to study disease progression or discover novel therapeutic or diagnostic markers in cancer research. In this chapter, we aim to introduce the cancer cell reprogramming protocol in detail by using human melanocytes and melanoma cell lines, and Sendai viral vectors encoding Yamanaka factors have been used to reprogram cells. Representative results are discussed and important notes have been summarized in order to point out important steps during cancer cell reprogramming.

Micro-RNA Profiling Reveals the Key Role of miR-206 in the Hematopoietic Potential of Human Embryonic and Induced Pluripotent Stem Cells

Although human pluripotent stem cells (hPSCs) can theoretically be differentiated into any cell type, their ability to generate hematopoietic cells shows a major variability from one cell line to another. The reasons of this variable differentiation potential, which is constant and reproducible in a given hPSC line, are not clearly established. In order to study this phenomenon, we comparatively studied 4 human embryonic stem cell lines (hESC) and 11 human induced pluripotent stem cell (hiPSC) lines using transcriptome assays. These cell lines exhibited a significant variability to generate in vitro hematopoiesis as evaluated by day-16 embryoid body (EB) formation followed by clonogenic (CFC) assays. Four out of 11 iPSC lines (PB6, PB9, PB12.1, and PB14.3) were found to lack any hematopoietic differentiation ability whereas 7 cell lines showed variable hematopoietic potential. Among hESC lines, H9 and CL0 had low H1 and SA01 exhibited high hematopoietic potential using the above assays. Among hESC and hIPSC displaying hematopoietic potential, two sub-groups were further defined based on their hematopoietic CFC efficiency: a group of poor (generation of less than 100 CFC/105 cells, PB4 / PB10 /H9 /CL01), and high hematopoietic competency (more than 120 CFC/105 cells, PB3/ PB6.1 /PB7 /PB13 /PB17 /SA01/H1). Using global miRNome analysis performed at the pluripotency stage, the expression of 754 individual miRNAs was analyzed from 15 hPSC lines in order to explore a potential predictive marker between both sub-groups of pluripotent cells according to their hematopoietic potency. Using this approach, 27 miRNAs out of 754 appeared differentially expressed allowing the identification of a miRNA signature associated with hematopoietic-competency. The hematopoietic competency was associated with down-regulation of miR-206, miR-135b, miR-105, miR-492, miR-622 and upregulation of miR-520a, miR-296, miR-122, miR-515, miR-335. Amongst these, miR-206 harbored the most significant variation (0.04-Fold change). To explore the role of miRNA-206 in this phenomenon, we have generated a miR-206-eFGP-Puro lentiviral vector which was transfected in hESC line H1 followed by puromycin selection. As a control, H1 cell line was transfected with a Arabidopsis thaliana microRNA sequence (ath-miR-159a), which has no specific targets in mammalian cells. The correct expression of the transgenes were evaluated by flow cytometry (using GFP) and q-RT-PCR for miR-206 expression. The hematopoietic potential of H1 cell line and its miR-206-overexpressing counterpart was then tested using standard in vitro assays via d16-EB generation. We found that both CFC numbers and percentage of CD34+ were significantly lower in H1-mir-206-derived day-16 EB cells than in H1-ath- derived day-16 EB cells (p < 0.05). Thus, over-expression of miR-206 in this blood-competent hESC appeared to repress its hematopoietic potential at very early stage, since a similar lower CFC efficiency was observed in day-3 EB cells derived from miR-206 overexpressing H1 cell line. We then conducted an integrative bioinformatics analysis on miR-206 predicted target genes. To this end, 773 mRNA target transcripts of the broadly conserved (across vertebrates) miR-1-3p/206 family were identified in the TargetScan database and were integrated into the global transcriptomic analysis performed by microarray on day-16 EB cells. Using supervised ranking product analysis, 62 predicted gene targets of the miR-1-3p/206 family were found to be significantly up-regulated in hematopoietic-competent EB samples including the transcription factors RUNX1 and TAL1. Hierarchical unsupervised clustering, based on this subset of 62 predicted mir-206 target genes, fully discriminated hematopoietic-deficient from hematopoietic-competent cells. In conclusion, miRNA profiling performed at pluripotency stage could be useful to predict the ability to human iPSC to give rise to blood cell progenitors. This work emphasizes for the first time the critical role of the muscle-specific miR-206 in hematopoietic differentiation. Finally, these results suggest that genetic manipulation of hESC/iPSC could be used to enhance their hematopoietic potential and to design protocols for generation of hPSC-derived hematopoietic stem cells with long-term reconstitution ability. DisclosuresNo relevant conflicts of interest to declare.

Generation and characterization of a human iPSC line from an ALS patient carrying the Q66K-MATR3 mutation

Fibroblasts isolated from an Amyotrophic Lateral Sclerosis (ALS)-patient carrying a mutation in Matrin-3 (p.Q66K -MATR3) gene were reprogrammed to the pluripotency stage by using non-integrating episomal plasmids. We generated the Q66K#44DRM induced pluripotent stem cell (iPSC) line that showed regular karyotype, expressed pluripotency-associated markers and were able to properly differentiate into the three germ layers. The heterozygous missense mutation in the MATR3 gene (p.Q66K), which is associated to ALS disease, was present in the generated iPSC line.Resource tableUnlabelled TableUnique stem cell line identifierCIBIOi001-AAlternative name(s) of stem cell lineQ66K#44DRMInstitutionCIBIO, University of Trento, ItalyContact information of distributorAlessandro Provenzani alessandro.provenzani@unitn.itType of cell lineiPSCs (induced pluripotent stem cells)OriginHumanAdditional origin infoAge:51Sex: MaleEthnicity if known: European, Italian.Cell sourcePatient derived fibroblastsClonalityClonal, clone number 44Method of reprogrammingEpisomal iPSC Reprogramming PlasmidGenetic modificationNOType of modificationSpontaneous mutation (missense)Associated diseaseAmyotrophic Lateral Sclerosis (ALS)Gene/locusMATR3/5q31.2Method of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock date16/01/2018Cell line repository/bankN/AEthical approvalPatient signed informed consent, number of protocol P/740/CE/2012 released by the ethical committee of the Catholic University School of Medicine

Abstract B41: Modeling bone development and cancer predisposition in Diamond Blackfan anemia (DBA)

Abstract DBA is an inherited bone marrow failure syndrome characterized by pure red cell aplasia, congenital physical anomalies, and cancer predisposition, in particular an increased incidence of osteogenic sarcoma (O:E 42.44). In order to investigate the mechanism of defective bone development and ultimately oncogenesis, we used an in vitro model consisting of mouse embryonic stem cells gene trapped for the Ribosomal Protein 19 gene (Rps19) and developed an in vivo transgenic conditional mouse model with floxed Rps19 alleles and a bone-specific expressing Col1a1-CreER. Using a 23-day stepwise osteoblast differentiation model, haploinsufficient Rps19+/- mouse ES cells did not show differences in the ability to develop into mesodermal progenitors based on qPCR analysis of mesodermal transcription factors, but displayed a decreased ability to produce mature osteoblasts based upon decreased alizarin red staining. In addition, higher levels of the Trp53 protein were found in Rps19+/- mouse ES cells at both the pluripotent and mesodermal stages of differentiation. Further, Rps19+/- mouse ES cells presented with aberrant WNT signaling, as represented by decreased levels of β-catenin early in osteoblast differentiation and rising to higher levels towards late osteoblast differentiation. We assessed the hypothesis of WNT signaling involvement in lineage specification and found that Rps19+/- mouse ES cell-derived osteoblasts show decreased levels of Runx2 and increased levels of Sox9. In the transgenic mouse model, tamoxifen-induced recombination of Rps19 at E15-17 did not show gross morphologic skeletal defects at E20.5 for Rps19fl/+ embryos, although Rps19fl/fl embryos displayed severe skeletal defects through alizarin red and alcian blue staining. In conclusion, haploinsufficient levels of Rps19 found in mice contribute to improper skeletal development and probably reflect the clinical scenario. We plan to utilize these in vitro and in vivo models to provide clues into the mechanism of ribosomal protein haploinsufficiency in tumor development, which may suggest surveillance and treatment strategies. Citation Format: Jimmy Hom, Brian Dulmovits, Luanne Peters, Adrianna Vlachos, Jeffrey M. Lipton, Lionel Blanc. Modeling bone development and cancer predisposition in Diamond Blackfan anemia (DBA) [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B41.

Transcriptional landscape changes during human embryonic stem cell derivation.

What are the transcriptional changes occurring during the human embryonic stem cell (hESC) derivation process, from the inner cell mass (ICM) to post-ICM intermediate stage (PICMI) to hESC stage, that have downstream effects on pluripotency states and differentiation? We reveal that although the PICMI is transcriptionally similar to the hESC profile and distinct from ICM, it exhibits upregulation of primordial germ cell (PGC) markers, dependence on leukemia inhibitory factor (LIF) signaling, upregulation of naïve pluripotency-specific signaling networks and appears to be an intermediate switching point from naïve to primed pluripotency. It is currently known that the PICMI exhibits markers of early and late-epiblast stage. It is suggested that hESCs acquire primed pluripotency features due to the upregulation of post-implantation genes in the PICMI which renders them predisposed towards differentiation cues. Despite this current knowledge, the transcriptional landscape changes during hESC derivation from ICM to hESC and the effect of PICMI on pluripotent state is still not well defined. To gain insight into the signaling mechanisms that may govern the ICM to PICMI to hESC transition, comparative RNA sequencing (RNA-seq) analysis was performed on preimplantation ICMs, PICMIs and hESCs in biological and technical triplicates (n = 3). Primed hESCs (XX) were maintained in feeder-free culture conditions on Matrigel for two passages and approximately 50 cells were collected in biological and technical triplicates (n = 3). For ICM sample collection, Day 3, frozen-thawed human embryos were cultured up to day five blastocyst stage and only good quality blastocysts were subjected to laser-assisted micromanipulation for ICM collection (n = 3). Next, day six expanded blastocysts were cultured on mouse embryonic fibroblasts and manual dissection was performed on the PICMI outgrowths between post-plating Day 6 and Day 10 (n = 3). Sequencing of these samples was performed on NextSeq500 and statistical analysis was performed using edgeR (false discovery rate (FDR) < 0.05). Comparative RNA-seq data analysis revealed that 634 and 560 protein-coding genes were significantly up and downregulated in hESCs compared to ICM (FDR < 0.05), respectively. Upon ICM to PICMI transition, 471 genes were expressed significantly higher in the PICMI compared to ICM, while 296 genes were elevated in the ICM alone (FDR < 0.05). Principle component analysis showed that the ICM was completely distinct from the PICMI and hESCs while the latter two clustered in close proximity to each other. Increased expression of E-CADHERIN1 (CDH1) in ICM and intermediate levels in the PICMI was observed, while CDH2 was higher in hESCs, suggesting a role of extracellular matrix components in facilitating pluripotency transition during hESC derivation. The PICMI also showed regulation of naïve-specific LIF and bone morphogenetic protein signaling, differential regulation of primed pluripotency-specific fibroblast growth factor and NODAL signaling pathway components, upregulation of phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway (PI3K/AKT/mTORC), as well as predisposition towards the germ cell lineage, further confirmed by gene ontology analysis. Hence, the data suggest that the PICMI may serve as an intermediate pluripotency stage which, when subjected to an appropriate culture niche, could aid in enhancing naïve hESC derivation and germ cell differentiation efficiency. Gene Expression Omnibus (GEO) Accession number GSE119378. Owing to the limitation in sample availability, the sex of ICM and PICMI have not been taken into consideration. Obtaining cells from the ICM and maintaining them in culture is not feasible as it will hamper the formation of PICMI and hESC derivation. Single-cell quantitative real-time PCR on low ICM and PICMI cell numbers, although challenging due to limited availability of human embryos, will be advantageous to further corroborate the RNA-seq data on transcriptional changes during hESC derivation process. We elucidate the dynamics of transcriptional network changes from the naïve ICM to the intermediate PICMI stage and finally the primed hESC lines. We provide an in-depth understanding of the PICMI and its role in conferring the type of pluripotent state which may have important downstream effects on differentiation, specifically towards the PGC lineage. This knowledge contributes to our limited understanding of the true nature of the human pluripotent state in vitro. This research is supported by the Concerted Research Actions funding from Bijzonder Onderzoeksfonds University Ghent (BOF GOA 01G01112).The authors declare no conflict of interest.

Transdifferentiation: a new promise for neurodegenerative diseases

Neurodegenerative diseases are characterized by a gradual loss of cognitive and physical functions. Medications for these disorders are limited and treat the symptoms only. There are no disease-modifying therapies available, which have been shown to slow or stop the continuing loss of neurons. Transdifferentiation, whereby somatic cells are reprogrammed into another lineage without going through an intermediate proliferative pluripotent stem cell stage, provides an alternative strategy for regenerative medicine and disease modeling. In particular, the transdifferentiation of somatic cells into specific subset of patient-specific neuronal cells offers alternative autologous cell therapeutic strategies for neurodegenerative disorders and presents a rich source of using diverse somatic cell types for relevant applications in translational, personalized medicine, as well as human mechanistic study, new drug-target identification, and novel drug screening systems. Here, we provide a comprehensive overview of the recent development of transdifferentiation research, with particular attention to chemical-induced transdifferentiation and perspectives for modeling and treatment of neurodegenerative diseases.

Abstract 4072: Direct transdifferentiation of glioblastoma cells to antigen-presenting cells: A novel immunotherapeutic approach

Abstract Cell-based immunotherapy has shown encouraging results in solid cancers. However, major limitations of this approach include difficulty with isolation and generation of effective antigen-presenting or immune effector cells and the high cost of cell-based vaccines. In glioblastoma (GBM), the most common and lethal brain cancer in adults, inadequate migration of immune cells across the blood-brain barrier represents another major obstacle to cell-based immunotherapy. One approach to overcome these barriers is to convert GBM cells directly into immune cells of interest. Transdifferentiation, the transformation of one cell type other than stem cells directly into another cell type without an intermediate pluripotent stage, has shown early promise in regenerative medicine, but has had a limited role as a cancer therapy, especially for solid tumors. This is in large part due to the laborious and time-consuming process of identifying master fate determinants of the cell types of interest in the transdifferentiation. To enable this novel application, we have developed and experimentally validated two tandem computational platforms, GeneRep and nSCORE. Applying this novel algorithm to large quantities of published and in-house gene expression datasets, we successfully predicted cell fate determinants of tens of different cell types with several having been validated in transdifferentiation experiments by others and us, such as the conversions of astrocytes to neuronal stem cells, of astrocytes to glioma stem-like cells, of fibroblasts to macrophages, etc. Here, we describe the process of combining a unique core set of hematopoietic stem cell, myeloid, and macrophage or dendritic cell (DC) fate determinants—some have not been described as critical in these cell fates—as predicted and highly ranked by GeneRep-nSCORE to efficiently transdifferentiate GBM cells into functional macrophages or dendritic cells, respectively, while neutralizing their malignant phenotype. This novel transdifferentiation has the potential for transformative impacts in many areas, including DC vaccine immunotherapy and cancer therapy in general, and pioneers the concept that solid tumor cells can be converted into another cell type, and not just any cell type, but one that potentially can elicit a therapeutic response against itself. Citation Format: Mathew Sebastian, Son B. Le, Changwang Deng, Nagheme Thomas, David D. Tran. Direct transdifferentiation of glioblastoma cells to antigen-presenting cells: A novel immunotherapeutic approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4072.

Reprogramming of Adult Peripheral Blood Cells into Human Induced Pluripotent Stem Cells as a Safe and Accessible Source of Endothelial Cells.

New approaches in regenerative medicine and vasculogenesis have generated a demand for sufficient numbers of human endothelial cells (ECs). ECs and their progenitors reside on the interior surface of blood and lymphatic vessels or circulate in peripheral blood; however, their numbers are limited, and they are difficult to expand after isolation. Recent advances in human induced pluripotent stem cell (hiPSC) research have opened possible avenues to generate unlimited numbers of ECs from easily accessible cell sources, such as the peripheral blood. In this study, we reprogrammed peripheral blood mononuclear cells, human umbilical vein endothelial cells (HUVECs), and human saphenous vein endothelial cells (HSVECs) into hiPSCs and differentiated them into ECs. The phenotype profiles, functionality, and genome stability of all hiPSC-derived ECs were assessed and compared with HUVECs and HSVECs. hiPSC-derived ECs resembled their natural EC counterparts, as shown by the expression of the endothelial surface markers CD31 and CD144 and the results of the functional analysis. Higher expression of endothelial progenitor markers CD34 and kinase insert domain receptor (KDR) was measured in hiPSC-derived ECs. An analysis of phosphorylated histone H2AX (γH2AX) foci revealed that an increased number of DNA double-strand breaks upon reprogramming into pluripotent cells. However, differentiation into ECs restored a normal number of γH2AX foci. Our hiPSCs retained a normal karyotype, with the exception of the HSVEC-derived hiPSC line, which displayed mosaicism due to a gain of chromosome 1. Peripheral blood from adult donors is a suitable source for the unlimited production of patient-specific ECs through the hiPSC interstage. hiPSC-derived ECs are fully functional and comparable to natural ECs. The protocol is eligible for clinical applications in regenerative medicine, if the genomic stability of the pluripotent cell stage is closely monitored.

Direct Neuronal Reprogramming for Disease Modeling Studies Using Patient-Derived Neurons: What Have We Learned?

Direct neuronal reprogramming, by which a neuron is formed via direct conversion from a somatic cell without going through a pluripotent intermediate stage, allows for the possibility of generating patient-derived neurons. A unique feature of these so-called induced neurons (iNs) is the potential to maintain aging and epigenetic signatures of the donor, which is critical given that many diseases of the CNS are age related. Here, we review the published literature on the work that has been undertaken using iNs to model human brain disorders. Furthermore, as disease-modeling studies using this direct neuronal reprogramming approach are becoming more widely adopted, it is important to assess the criteria that are used to characterize the iNs, especially in relation to the extent to which they are mature adult neurons. In particular: i) what constitutes an iN cell, ii) which stages of conversion offer the earliest/optimal time to assess features that are specific to neurons and/or a disorder and iii) whether generating subtype-specific iNs is critical to the disease-related features that iNs express. Finally, we discuss the range of potential biomedical applications that can be explored using patient-specific models of neurological disorders with iNs, and the challenges that will need to be overcome in order to realize these applications.

Direct reprogramming from fibroblasts into cardiamyocytes

Cardiac regenerative therapy has attracted much attention as a novel approach for heart diseases. Direct reprogramming of fibroblasts into cardiomyocytes without going through a pluripotent stem cell stage would provide a promising source of cells for cell transplantation in future. This review summarizes the research methods and problems of direct reprogramming of fibroblasts into cardiomyocytes in vitro and in vivo, and forecasts the future development of this new strategy.

Simple Maturation of Direct-Converted Hepatocytes Derived from Fibroblasts.

Target cells differentiation techniques from stem cells are developed rapidly. Recently, direct conversion techniques are introduced in various categories. Unlike pluripotent stem cells, this technique enables direct differentiation into the other cell types such as neurons, cardiomyocytes, insulin-producing cells, and hepatocytes without going through the pluripotent stage. However, the function of these converted cells reserve an immature phenotype. Therefore, we modified the culture conditions of mouse direct converted hepatocytes (miHeps) to mature fetal characteristics, such as higher AFP and lower albumin (ALB) expression than primary hepatocytes. First, we generate miHeps from mouse embryonic fibroblasts (MEFs) with two transcription factors HNF4α and Foxa3. These cells indicate typical epithelial morphology and express hepatic proteins. To mature hepatic function, DMSO is treated during culture time for more than 7days. After maturation, miHeps showed features of maturation such as exhibiting typical hepatocyte-like morphology, increased up-regulated ALB and CYP enzyme gene expression, down-regulated AFP expressions, and acquired hepatic function over time. Thus, our data provides a simple method to mature direct converted hepatocytes functionally and these cells enable them to move closer to generating functional hepatocytes.

Engineering kidney cells: reprogramming and directed differentiation to renal tissues.

Growing knowledge ofhow cell identity is determined at the molecular level has enabled the generation of diverse tissue types, including renal cells from pluripotent or somatic cells. Recently, several in vitro protocols involving either directed differentiation or transcription-factor-based reprogramming to kidney cells have been established. Embryonic stem cells or induced pluripotent stem cells can be guided towards a kidney fate by exposing them to combinations of growth factors or small molecules. Here, renal development is recapitulated in vitro resulting in kidney cells or organoids that show striking similarities to mammalian embryonic nephrons. In addition, culture conditions are also defined that allow the expansion of renal progenitor cells in vitro. Another route towards the generation of kidney cells is direct reprogramming. Key transcription factors are used to directly impose renal cell identity on somatic cells, thus circumventing the pluripotent stage. This complementary approach to stem-cell-based differentiation has been demonstrated to generate renal tubule cells and nephron progenitors. In-vitro-generated renal cells offer new opportunities for modelling inherited and acquired renal diseases on a patient-specific genetic background. These cells represent a potential source for developing novel models for kidney diseases, drug screening and nephrotoxicity testing and might represent the first steps towards kidney cell replacement therapies. In this review, we summarize current approaches for the generation of renal cells in vitro and discuss the advantages of each approach and their potential applications.

Cytokine-free directed differentiation of human pluripotent stem cells efficiently produces hemogenic endothelium with lymphoid potential

BackgroundThe robust generation of human hematopoietic progenitor cells from induced or embryonic pluripotent stem cells would be beneficial for multiple areas of research, including mechanistic studies of hematopoiesis, the development of cellular therapies for autoimmune diseases, induced transplant tolerance, anticancer immunotherapies, disease modeling, and drug/toxicity screening. Over the past years, significant progress has been made in identifying effective protocols for hematopoietic differentiation from pluripotent stem cells and understanding stages of mesodermal, endothelial, and hematopoietic specification. Thus, it has been shown that variations in cytokine and inhibitory molecule treatments in the first few days of hematopoietic differentiation define primitive versus definitive potential of produced hematopoietic progenitor cells. The majority of current feeder-free, defined systems for hematopoietic induction from pluripotent stem cells include prolonged incubations with various cytokines that make the differentiation process complex and time consuming. We established that the application of Wnt agonist CHIR99021 efficiently promotes differentiation of human pluripotent stem cells in the absence of any hematopoietic cytokines to the stage of hemogenic endothelium capable of definitive hematopoiesis.MethodsThe hemogenic endothelium differentiation was accomplished in an adherent, serum-free culture system by applying CHIR99021. Hemogenic endothelium progenitor cells were isolated on day 5 of differentiation and evaluated for their endothelial, myeloid, and lymphoid potential.ResultsMonolayer induction based on GSK3 inhibition, described here, yielded a large number of CD31+CD34+ hemogenic endothelium cells. When isolated and propagated in adherent conditions, these progenitors gave rise to mature endothelium. When further cocultured with OP9 mouse stromal cells, these progenitors gave rise to various cells of myeloid lineages as well as natural killer lymphoid, T-lymphoid, and B-lymphoid cells.ConclusionThe results of this study substantiate a method that significantly reduces the complexity of current protocols for hematopoietic induction, offers a defined system to study the factors that affect the early stages of hematopoiesis, and provides a new route of lymphoid and myeloid cell derivation from human pluripotent stem cells, thus enhancing their use in translational medicine.

Glycoconjugates reveal diversity of human neural stem cells (hNSCs) derived from human induced pluripotent stem cells (hiPSCs).

Neural stem cells (NSCs) have the ability to self-renew and to differentiate into various cell types of the central nervous system. This potential can be recapitulated by human induced pluripotent stem cells (hiPSCs) in vitro. The differentiation capacity of hiPSCs is characterized by several stages with distinct morphologies and the expression of various marker molecules. We used the monoclonal antibodies (mAbs) 487LeX, 5750LeX and 473HD to analyze the expression pattern of particular carbohydrate motifs as potential markers at six differentiation stages of hiPSCs. Mouse ESCs were used as a comparison. At the pluripotent stage, 487LeX-, 5750LeX- and 473HD-related glycans were differently expressed. Later, cells of the three germ layers in embryoid bodies (hEBs) and, even after neuralization of hEBs, subpopulations of cells were labeled with these surface antibodies. At the human rosette-stage of NSCs (hR-NSC), LeX- and 473HD-related epitopes showed antibody-specific expression patterns. We also found evidence that these surface antibodies could be used to distinguish the hR-NSCs from the hSR-NSCs stages. Characterization of hNSCsFGF-2/EGF derived from hSR-NSCs revealed that both LeX antibodies and the 473HD antibody labeled subpopulations of hNSCsFGF-2/EGF. Finally, we identified potential LeX carrier molecules that were spatiotemporally regulated in early and late stages of differentiation. Our study provides new insights into the regulation of glycoconjugates during early human stem cell development. The mAbs 487LeX, 5750LeX and 473HD are promising tools for identifying distinct stages during neural differentiation.

The gastrula transition reorganizes replication-origin selection in Caenorhabditis elegans.

Although some features underlying replication-origin activation in metazoan cells have been determined, little is known about their regulation during metazoan development. Using the nascent-strand purification method, here we identified replication origins throughout Caenorhabditis elegans embryonic development and found that the origin repertoire is thoroughly reorganized after gastrulation onset. During the pluripotent embryonic stages (pregastrula), potential cruciform structures and open chromatin are determining factors that establish replication origins. The observed enrichment of replication origins in transcription factor-binding sites and their presence in promoters of highly transcribed genes, particularly operons, suggest that transcriptional activity contributes to replication initiation before gastrulation. After the gastrula transition, when embryonic differentiation programs are set, new origins are selected at enhancers, close to CpG-island-like sequences, and at noncoding genes. Our findings suggest that origin selection coordinates replication initiation with transcriptional programs during metazoan development.