A Drug-Inducible System for Direct Reprogramming of Human Somatic Cells to Pluripotency

SummaryCurrent approaches to reprogram human somatic cells to pluripotent iPSCs utilize viral transduction of different combinations of transcription factors. These protocols are highly inefficient because only a small fraction of cells carry the appropriate number and stoichiometry of proviral insertions to initiate the reprogramming process. Here we have generated genetically homogeneous “secondary” somatic cells, which carry the reprogramming factors as defined doxycycline (DOX)-inducible transgenes. These cells were obtained by infecting fibroblasts with DOX-inducible lentiviruses, isolating “primary” iPSCs in the presence of the drug, and finally differentiating to “secondary” fibroblasts. When “secondary” fibroblast lines were cultured in the presence of DOX without further viral infection, up to 2% of the cells were reprogrammed to pluripotent “secondary” human iPSCs. This system will facilitate the characterization of the reprogramming process and provides a unique platform for genetic or chemical screens to enhance reprogramming or replace individual factors

Lectin binding profiles of SSEA-4 enriched, pluripotent human embryonic stem cell surfaces

BACKGROUND: Pluripotent human embryonic stem cells (hESCs) have the potential to form every cell type in the body. These cells must be appropriately characterized prior to differentiation studies or when defining characteristics of the pluripotent state. Some developmentally regulated cell surface antigens identified by monoclonal antibodies in a variety of species and stem cell types have proven to be side chains of membrane glycolipids and glycoproteins. Therefore, to examine hESC surfaces for other potential pluripotent markers, we used a panel of 14 lectins, which were chosen based on their specificity for a variety of carbohydrates and carbohydrate linkages, along with stage specific embryonic antigen-4 (SSEA-4), to determine binding quantitation by flow cytometry and binding localization in adherent colonies by immunocytochemistry. RESULTS: Enriching cells for SSEA-4 expression increased the percentage of SSEA-4 positive cells to 98–99%. Using enriched high SSEA-4-expressing hESCs, we then analyzed the binding percentages of selected lectins and found a large variation in binding percentages ranging from 4% to 99% binding. Lycopersicon (tomato)esculetum lectin (TL), Ricinus communis agglutinin (RCA), and Concanavalin A (Con A) bound to SSEA-4 positive regions of hESCs and with similar binding percentages as SSEA-4. In contrast, we found Dolichos biflorus agglutinin (DBA) and Lotus tetragonolobus lectin (LTL) did not bind to hESCs while Phaseolus vulgaris leuco-agglutinin (PHA-L), Vicia villosa agglutinin (VVA), Ulex europaeus agglutinin (UEA), Phaseolus vulgaris erythro-agglutinin (PHA-E), and Maackia amurensis agglutinin (MAA) bound partially to hESCs. These binding percentages correlated well with immunocytochemistry results. CONCLUSION: Our results provide information about types of carbohydrates and carbohydrate linkages found on pluripotent hESC surfaces. We propose that TL, RCA and Con A may be used as markers that are associated with the pluripotent state of hESCs because binding percentages and binding localization of these lectins are similar to those of SSEA-4. Non-binding lectins, DBA and LTL, may identify differentiated cell types; however, we did not find these lectins to bind to pluripotent SSEA-4 positive hESCs. This work represents a fundamental base to systematically classify pluripotent hESCs, and in future studies these lectins may be used to distinguish differentiated hESC types based on glycan presentation that accompanies differentiation

Chromatin Structure and Gene Expression Programs of Human Embryonic and Induced Pluripotent Stem Cells

Knowledge of both the global chromatin structure and the gene expression programs of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) should provide a robust means to assess whether the genomes of these cells have similar pluripotent states. Recent studies have suggested that ESCs and iPSCs represent different pluripotent states with substantially different gene expression profiles. We describe here a comparison of global chromatin structure and gene expression data for a panel of human ESCs and iPSCs. Genome-wide maps of nucleosomes with histone H3K4me3 and H3K27me3 modifications indicate that there is little difference between ESCs and iPSCs with respect to these marks. Gene expression profiles confirm that the transcriptional programs of ESCs and iPSCs show very few consistent differences. Although some variation in chromatin structure and gene expression was observed in these cell lines, these variations did not serve to distinguish ESCs from iPSCs

Efficient derivation of microglia-like cells from human pluripotent stem cells

Microglia, the only lifelong resident immune cells of the central nervous system (CNS), are highly specialized macrophages that have been recognized to have a crucial role in neurodegenerative diseases such as Alzheimer's, Parkinson's and adrenoleukodystrophy (ALD). However, in contrast to other cell types of the human CNS, bona fide microglia have not yet been derived from cultured human pluripotent stem cells. Here we establish a robust and efficient protocol for the rapid production of microglia-like cells from human (h) embryonic stem (ES) and induced pluripotent stem (iPS) cells that uses defined serum-free culture conditions. These in vitro pluripotent stem cell-derived microglia-like cells (termed pMGLs) faithfully recapitulate the expected ontogeny and characteristics of their in vivo counterparts, and they resemble primary fetal human and mouse microglia. We generated these cells from multiple disease-specific cell lines and find that pMGLs derived from an hES model of Rett syndrome are smaller than their isogenic controls. We further describe a platform to study the integration and live behavior of pMGLs in organotypic 3D cultures. This modular differentiation system allows for the study of microglia in highly defined conditions as they mature in response to developmentally relevant cues, and it provides a framework in which to study the long-term interactions of microglia residing in a tissue-like environment.Simons Foundation (Grant SFARI 204106)National Institutes of Health (U.S.) (Grant HD 045022)National Institutes of Health (U.S.) (Grant R37-CA084198)National Institutes of Health (U.S.) (Grant NS088538)National Institutes of Health (U.S.) (Grant 1RF1 AG042978

Derivation of Pre-X Inactivation Human Embryonic Stem Cells under Physiological Oxygen Concentrations

The presence of two active X chromosomes (XaXa) is a hallmark of the ground state of pluripotency specific to murine embryonic stem cells (ESCs). Human ESCs (hESCs) invariably exhibit signs of X chromosome inactivation (XCI) and are considered developmentally more advanced than their murine counterparts. We describe the establishment of XaXa hESCs derived under physiological oxygen concentrations. Using these cell lines, we demonstrate that (1) differentiation of hESCs induces random XCI in a manner similar to murine ESCs, (2) chronic exposure to atmospheric oxygen is sufficient to induce irreversible XCI with minor changes of the transcriptome, (3) the Xa exhibits heavy methylation of the XIST promoter region, and (4) XCI is associated with demethylation and transcriptional activation of XIST along with H3K27-me3 deposition across the Xi. These findings indicate that the human blastocyst contains pre-X-inactivation cells and that this state is preserved in vitro through culture under physiological oxygen.Susan WhiteheadHillel and Liliana Bachrac

Systematic Identification of Culture Conditions for Induction and Maintenance of Naive Human Pluripotency

Embryonic stem cells (ESCs) of mice and humans have distinct molecular and biological characteristics, raising the question of whether an earlier, “naive” state of pluripotency may exist in humans. Here we took a systematic approach to identify small molecules that support self-renewal of naive human ESCs based on maintenance of endogenous OCT4 distal enhancer activity, a molecular signature of ground state pluripotency. Iterative chemical screening identified a combination of five kinase inhibitors that induces and maintains OCT4 distal enhancer activity when applied directly to conventional human ESCs. These inhibitors generate human pluripotent cells in which transcription factors associated with the ground state of pluripotency are highly upregulated and bivalent chromatin domains are depleted. Comparison with previously reported naive human ESCs indicates that our conditions capture a distinct pluripotent state in humans that closely resembles that of mouse ESCs. This study presents a framework for defining the culture requirements of naive human pluripotent cells.Simons Foundation (Grant SFLIFE 286977)National Institutes of Health (U.S.) (Grant RO1-CA084198)National Science Foundation (U.S.). Graduate Research FellowshipJerome and Florence Brill Graduate Student Fellowshi

Human embryonic stem cells from aneuploid blastocysts identified by pre-implantation genetic screening

Human embryonic stem cells are derived from the inner cell mass of pre-implantation embryos. The cells have unlimited proliferation potential and capacity to differentiate into the cells of the three germ layers. Human embryonic stem cells are used to study human embryogenesis and disease modeling and may in the future serve as cells for cell therapy and drug screening. Human embryonic stem cells are usually isolated from surplus normal frozen embryos and were suggested to be isolated from diseased embryos detected by pre-implantation genetic diagnosis. Here we report the isolation of 12 human embryonic stem cell lines and their thorough characterization. The lines were derived from embryos detected to have aneuploidy by pre-implantation genetic screening. Karyotype analysis of these cell lines showed that they are euploid, having 46 chromosomes. Our interpretation is that the euploid cells originated from mosaic embryos, and in vitro selection favored the euploid cells. The undifferentiated cells exhibited long-term proliferation and expressed markers typical for embryonic stem cells such as OCT4, NANOG, and TRA-1-60. The cells manifested pluripotent differentiation both in vivo and in vitro. To further characterize the different lines, we have analyzed their ethnic origin and the family relatedness among them. The above results led us to conclude that the aneuploid mosaic embryos that are destined to be discarded can serve as source for normal euploid human embryonic stem cell lines. These lines represent various ethnic groups; more lines are needed to represent all populations

Metastable Pluripotent States in NOD Mouse Derived ES Cells

Embryonic stem (ES) cells are isolated from the inner cell mass (ICM) of blastocysts, whereas epiblast stem cells (EpiSCs) are derived from the post-implantation epiblast and display a restricted developmental potential. Here we characterize pluripotent states in the non-obese diabetic (NOD) mouse strain, which prior to this study was considered “non-permissive” for ES cell derivation. We find that NOD stem cells can be stabilized by providing constitutive expression of Klf4 or c-Myc or small molecules that can replace these factors during in vitro reprogramming. The NOD ES and iPS cells appear “metastable”, as they acquire an alternative EpiSC-like identity after removal of the exogenous factors, while their reintroduction converts the cells back to ICM-like pluripotency. Our findings suggest that stem cells from different genetic backgrounds can assume distinct states of pluripotency in vitro, the stability of which is regulated by endogenous genetic determinants and can be modified by exogenous factors.National Institutes of Health (U.S.) (Grant RO1-HDO45022)National Institutes of Health (U.S.) (Grant R37-CA084198)National Institutes of Health (U.S.) (Grant RO1-CA087869

Parkinson's Disease Patient-Derived Induced Pluripotent Stem Cells Free of Viral Reprogramming Factors

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may provide a source for replacement therapies. However, the use of viruses encoding the reprogramming factors represents a major limitation of the current technology since even low vector expression may alter the differentiation potential of the iPSCs or induce malignant transformation. Here, we show that fibroblasts from five patients with idiopathic Parkinson's disease can be efficiently reprogrammed and subsequently differentiated into dopaminergic neurons. Moreover, we derived hiPSCs free of reprogramming factors using Cre-recombinase excisable viruses. Factor-free hiPSCs maintain a pluripotent state and show a global gene expression profile, more closely related to hESCs than to hiPSCs carrying the transgenes. Our results indicate that residual transgene expression in virus-carrying hiPSCs can affect their molecular characteristics and that factor-free hiPSCs therefore represent a more suitable source of cells for modeling of human disease.Howard Hughes Medical Institute (Collaborative Innovation Award)Life Sciences Research Foundation (Merck Fellow)Michael Stern Parkinson's Research FoundationMorris K. Udall Center for Excellence in Parkinson’s Research (grant P50NS39793)National Institutes of Health (U.S.) (NIH grant R37-CA084198)National Institutes of Health (U.S.) (NIH grant RO1-CA087869)National Institutes of Health (U.S.) (grant NIH RO1-HD045022

Normal Human Pluripotent Stem Cell Lines Exhibit Pervasive Mosaic Aneuploidy

Human pluripotent stem cell (hPSC) lines have been considered to be homogeneously euploid. Here we report that normal hPSC – including induced pluripotent - lines are karyotypic mosaics of euploid cells intermixed with many cells showing non-clonal aneuploidies as identified by chromosome counting, spectral karyotyping (SKY) and fluorescent in situ hybridization (FISH) of interphase/non-mitotic cells. This mosaic aneuploidy resembles that observed in progenitor cells of the developing brain and preimplantation embryos, suggesting that it is a normal, rather than pathological, feature of stem cell lines. The karyotypic heterogeneity generated by mosaic aneuploidy may contribute to the reported functional and phenotypic heterogeneity of hPSCs lines, as well as their therapeutic efficacy and safety following transplantation