Epigenetic dynamics during capacitation of naïve human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) are of fundamental relevance in regenerative medicine. Naïve hPSCs hold promise to overcome some of the limitations of conventional (primed) hPSCs, including recurrent epigenetic anomalies. Naïve-to-primed transition (capacitation) follows transcriptional dynamics of human embryonic epiblast and is necessary for somatic differentiation from naïve hPSCs. We found that capacitated hPSCs are transcriptionally closer to postimplantation epiblast than conventional hPSCs. This prompted us to comprehensively study epigenetic and related transcriptional changes during capacitation. Our results show that CpG islands, gene regulatory elements, and retrotransposons are hotspots of epigenetic dynamics during capacitation and indicate possible distinct roles of specific epigenetic modifications in gene expression control between naïve and primed hPSCs. Unexpectedly, PRC2 activity appeared to be dispensable for the capacitation. We find that capacitated hPSCs acquire an epigenetic state similar to conventional hPSCs. Significantly, however, the X chromosome erosion frequently observed in conventional female hPSCs is reversed by resetting and subsequent capacitation.

NANOGP1, a tandem duplicate of NANOG, exhibits partial functional conservation in human naïve pluripotent stem cells.

Gene duplication events can drive evolution by providing genetic material for new gene functions, and they create opportunities for diverse developmental strategies to emerge between species. To study the contribution of duplicated genes to human early development, we examined the evolution and function of NANOGP1, a tandem duplicate of the transcription factor NANOG. We found that NANOGP1 and NANOG have overlapping but distinct expression profiles, with high NANOGP1 expression restricted to early epiblast cells and naïve-state pluripotent stem cells. Sequence analysis and epitope-tagging revealed that NANOGP1 is protein coding with an intact homeobox domain. The duplication that created NANOGP1 occurred earlier in primate evolution than previously thought and has been retained only in great apes, whereas Old World monkeys have disabled the gene in different ways, including homeodomain point mutations. NANOGP1 is a strong inducer of naïve pluripotency; however, unlike NANOG, it is not required to maintain the undifferentiated status of human naïve pluripotent cells. By retaining expression, sequence and partial functional conservation with its ancestral copy, NANOGP1 exemplifies how gene duplication and subfunctionalisation can contribute to transcription factor activity in human pluripotency and development.

Amniogenesis occurs in two independent waves in primates.

In primates, the amnion emerges through cavitation of the epiblast during implantation, whereas in other species it does so later at gastrulation by the folding of the ectoderm. How the mechanisms of amniogenesis diversified during evolution remains unknown. Unexpectedly, single-cell analysis of primate embryos uncovered two transcriptionally and temporally distinct amniogenesis waves. To study this, we employed the naive-to-primed transition of human pluripotent stem cells (hPSCs) to model peri-implantation epiblast development. Partially primed hPSCs transiently gained the ability to differentiate into cavitating epithelium that transcriptionally and morphologically matched the early amnion, whereas fully primed hPSCs produced cells resembling the late amnion instead, thus recapitulating the two independent differentiation waves. The early wave follows a trophectoderm-like pathway and encompasses cavitation, whereas the late wave resembles an ectoderm-like route during gastrulation. The discovery of two independent waves explains how amniogenesis through cavitation could emerge during evolution via duplication of the pre-existing trophectoderm program.

8C-like cells capture the human zygotic genome activation program in vitro.

The activation of the embryonic genome marks the first major wave of transcription in the developing organism. Zygotic genome activation (ZGA) in mouse 2-cell embryos and 8-cell embryos in humans is crucial for development. Here, we report the discovery of human 8-cell-like cells (8CLCs) among naive embryonic stem cells, which transcriptionally resemble the 8-cell human embryo. They express ZGA markers, including ZSCAN4 and LEUTX, and transposable elements, such as HERVL and MLT2A1. 8CLCs show reduced SOX2 levels and can be identified using TPRX1 and H3.Y marker proteins in vitro. Overexpression of the transcription factor DUX4 and spliceosome inhibition increase human ZGA-like transcription. Excitingly, the 8CLC markers TPRX1 and H3.Y are also expressed in ZGA-stage 8-cell human embryos and may thus be relevant in vivo. 8CLCs provide a unique opportunity to characterize human ZGA-like transcription and might provide critical insights into early events in embryogenesis in humans.

Maintenance of Human Naïve Pluripotent Stem Cells.

Naïve pluripotent stem cells are the in vitro counterparts of pre-implantation embryonic epiblast. During the last few years, several protocols for establishing and maintaining human pluripotent stem cells (hPSCs) with naïve features have been reported, and many of these protocols result in cell populations with different molecular characteristics. As such, choosing the most appropriate method for naïve hPSC maintenance can pose a significant challenge. This chapter presents an optimized system called PXGL for culturing naïve hPSCs. Naïve hPSCs robustly self-renew while retaining a normal karyotype in PXGL, and the protocol is reproducible across different cell lines and independent laboratories.

Capacitation of Human Naïve Pluripotent Stem Cells.

Naïve and primed pluripotent stem cells resemble epiblast cells of the pre-implantation and post-implantation embryo, respectively. This chapter describes a simple experimental system for the efficient and consistent transition of human pluripotent stem cells (hPSCs) from the naïve to the primed state, which is a process called capacitation. Naïve hPSCs after capacitation can be differentiated further to somatic lineages, thus reproducing the order of developmental events in the embryo. Protocols for the induction of neuroectoderm, definitive endoderm, and paraxial mesoderm from hPSCs after capacitation and also from conventionally derived primed hPSCs are included in the chapter. Importantly, hPSC capacitation closely recapitulates transcriptional, metabolic, signaling, and cell polarity changes in the epiblast of primate embryos, and therefore offers a unique in vitro model of human peri-implantation development.

Profiling of naïve and primed human pluripotent stem cells reveals state-associated miRNAs.

Naïve human pluripotent stem cells (hPSC) resemble the embryonic epiblast at an earlier time-point in development than conventional, 'primed' hPSC. We present a comprehensive miRNA profiling of naïve-to-primed transition in hPSC, a process recapitulating aspects of early in vivo embryogenesis. We identify miR-143-3p and miR-22-3p as markers of the naïve state and miR-363-5p, several members of the miR-17 family, miR-302 family as primed markers. We uncover that miR-371-373 are highly expressed in naïve hPSC. MiR-371-373 are the human homologs of the mouse miR-290 family, which are the most highly expressed miRNAs in naïve mouse PSC. This aligns with the consensus that naïve hPSC resemble mouse naive PSC, showing that the absence of miR-371-373 in conventional hPSC is due to cell state rather than a species difference.

Wnt Inhibition Facilitates RNA-Mediated Reprogramming of Human Somatic Cells to Naive Pluripotency.

In contrast to conventional human pluripotent stem cells (hPSCs) that are related to post-implantation embryo stages, naive hPSCs exhibit features of pre-implantation epiblast. Naive hPSCs are established by resetting conventional hPSCs, or are derived from dissociated embryo inner cell masses. Here we investigate conditions for transgene-free reprogramming of human somatic cells to naive pluripotency. We find that Wnt inhibition promotes RNA-mediated induction of naive pluripotency. We demonstrate application to independent human fibroblast cultures and endothelial progenitor cells. We show that induced naive hPSCs can be clonally expanded with a diploid karyotype and undergo somatic lineage differentiation following formative transition. Induced naive hPSC lines exhibit distinctive surface marker, transcriptome, and methylome properties of naive epiblast identity. This system for efficient, facile, and reliable induction of transgene-free naive hPSCs offers a robust platform, both for delineation of human reprogramming trajectories and for evaluating the attributes of isogenic naive versus conventional hPSCs.

Prospective isolation of nonhematopoietic cells of the niche and their differential molecular interactions with HSCs.

A major limitation preventing in vivo modulation of hematopoietic stem cells (HSCs) is the incomplete understanding of the cellular and molecular support of the microenvironment in regulating HSC fate decisions. Consequently, murine HSCs cannot be generated, maintained, or expanded in culture over extended periods of time. A significantly improved understanding of the bone marrow niche environment and its molecular interactions with HSCs is pivotal to overcoming this challenge. We here prospectively isolated all major nonhematopoietic cellular niche components and cross-correlate them in detail with niche cells defined by lineage marking or tracing. Compiling an extensive database of soluble and membrane-bound ligand-receptor interactions, we developed a computational method to infer potential cell-to-cell interactions based on transcriptome data of sorter-purified niche cells and hematopoietic stem and progenitor cell subpopulations. Thus, we establish a compendium of the molecular communication between defined niche components and HSCs. Our analysis suggests an important role for cytokine antagonists in the regulation of HSC functions.

Capacitation of human naïve pluripotent stem cells for multi-lineage differentiation.

Human naïve pluripotent stem cells (PSCs) share features with the pre-implantation epiblast. They therefore provide an unmatched opportunity for characterising the developmental programme of pluripotency in Homo sapiens Here, we confirm that naïve PSCs do not respond directly to germ layer induction, but must first acquire competence. Capacitation for multi-lineage differentiation occurs without exogenous growth factor stimulation and is facilitated by inhibition of Wnt signalling. Whole-transcriptome profiling during this formative transition highlights dynamic changes in gene expression, which affect many cellular properties including metabolism and epithelial features. Notably, naïve pluripotency factors are exchanged for postimplantation factors, but competent cells remain devoid of lineage-specific transcription. The gradual pace of transition for human naïve PSCs is consistent with the timespan of primate development from blastocyst to gastrulation. Transcriptome trajectory during in vitro capacitation of human naïve cells tracks the progression of the epiblast during embryogenesis in Macaca fascicularis, but shows greater divergence from mouse development. Thus, the formative transition of naïve PSCs in a simple culture system may recapitulate essential and specific features of pluripotency dynamics during an inaccessible period of human embryogenesis.

Clonal Analysis Delineates Transcriptional Programs of Osteogenic and Adipogenic Lineages of Adult Mouse Skeletal Progenitors.

Bone, cartilage, and marrow adipocytes are generated by skeletal progenitors, but the relationships between lineages and mechanisms controlling their differentiation are poorly understood. We established mouse clonal skeletal progenitors with distinct differentiation properties and analyzed their transcriptome. Unipotent osteogenic and adipogenic cells expressed specific transcriptional programs, whereas bipotent clones combined expression of those genes and did not show a unique signature. We tested potential regulators of lineage commitment and found that in the presence of interferon-γ (IFNγ) adipogenic clones can be induced to osteogenesis and that their adipogenic capacity is inhibited. Analysis of IFNγ-regulated genes showed that lineage signatures and fate commitment of skeletal progenitors were controlled by EGR1 and EGR2. Knockdown experiments revealed that EGR1 is a positive regulator of the adipogenic transcriptional program and differentiation capacity, whereas EGR2 inhibits the osteogenic program and potency. Therefore, our work revealed transcriptional signatures of osteogenic and adipogenic lineages and mechanism triggering cell fate.

Epigenetic resetting of human pluripotency.

Much attention has focussed on the conversion of human pluripotent stem cells (PSCs) to a more naïve developmental status. Here we provide a method for resetting via transient histone deacetylase inhibition. The protocol is effective across multiple PSC lines and can proceed without karyotype change. Reset cells can be expanded without feeders with a doubling time of around 24 h. WNT inhibition stabilises the resetting process. The transcriptome of reset cells diverges markedly from that of primed PSCs and shares features with human inner cell mass (ICM). Reset cells activate expression of primate-specific transposable elements. DNA methylation is globally reduced to a level equivalent to that in the ICM and is non-random, with gain of methylation at specific loci. Methylation imprints are mostly lost, however. Reset cells can be re-primed to undergo tri-lineage differentiation and germline specification. In female reset cells, appearance of biallelic X-linked gene transcription indicates reactivation of the silenced X chromosome. On reconversion to primed status, XIST-induced silencing restores monoallelic gene expression. The facile and robust conversion routine with accompanying data resources will enable widespread utilisation, interrogation, and refinement of candidate naïve cells.

Retinal Organoids from Pluripotent Stem Cells Efficiently Recapitulate Retinogenesis.

The plasticity of pluripotent stem cells provides new possibilities for studying development, degeneration, and regeneration. Protocols for the differentiation of retinal organoids from embryonic stem cells have been developed, which either recapitulate complete eyecup morphogenesis or maximize photoreceptor genesis. Here, we have developed a protocol for the efficient generation of large, 3D-stratified retinal organoids that does not require evagination of optic-vesicle-like structures, which so far limited the organoid yield. Analysis of gene expression in individual organoids, cell birthdating, and interorganoid variation indicate efficient, reproducible, and temporally regulated retinogenesis. Comparative analysis of a transgenic reporter for PAX6, a master regulator of retinogenesis, shows expression in similar cell types in mouse in vivo, and in mouse and human retinal organoids. Early or late Notch signaling inhibition forces cell differentiation, generating organoids enriched with cone or rod photoreceptors, respectively, demonstrating the power of our improved organoid system for future research in stem cell biology and regenerative medicine.

Functional Restoration of gp91phox-Oxidase Activity by BAC Transgenesis and Gene Targeting in X-linked Chronic Granulomatous Disease iPSCs.

Chronic granulomatous disease (CGD) is an inherited immunodeficiency, caused by the inability of neutrophils to produce functional NADPH oxidase required for fighting microbial infections. The X-linked form of CGD (X-CGD), which is due to mutations in the CYBB (gp91phox) gene, a component of NADPH oxidase, accounts for about two-thirds of CGD cases. We derived induced pluripotent stem cells (iPSCs) from X-CGD patient keratinocytes using a Flp recombinase excisable lentiviral reprogramming vector. For restoring gp91phox function, we applied two strategies: transposon-mediated bacterial artificial chromosome (BAC) transgenesis and gene targeting using vectors with a fixed 5' homology arm (HA) of 8 kb and 3'HA varying in size from 30 to 80 kb. High efficiency of homologous recombination (up to 22%) was observed with increased size of the 3'HA. Both, BAC transgenesis and gene targeting resulted in functional restoration of the gp91phox measured by an oxidase activity assay in X-CGD iPSCs differentiated into the myeloid lineage. In conclusion, we delivered an important milestone towards the use of genetically corrected autologous cells for the treatment of X-CGD and monogenic diseases in general.

Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells.

Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification.

Transposon mediated BAC transgenesis via pronuclear injection of mouse zygotes.

Pronuclear microinjection of bacterial artificial chromosomes (BACs) is the preferred way to generate transgenic mice because the transgene accurately recapitulates expression of the endogenous gene. However, the method is demanding and the integrity and copy number of the BAC transgene is difficult to control. Here, we describe a simpler pronuclear injection method that relies on transposition to introduce full-length BACs into the mouse genome. The bacterial backbone of a hPAX6-GFP reporter BAC was retrofitted with PiggyBac transposon inverted terminal repeats and co-injected with PiggyBac transposase mRNA. Both the frequency of transgenic founders as well as intact, full-length, single copy integrations were increased. Transposition was determined by a rapid PCR screen for a transpositional signature and confirmation by splinkerette sequencing to show that the BACs were integrated as a single copy either in one or two different genomic sites. BAC transposons displayed improved functional accuracy over random integrants as evaluated by expression of the hPAX6-GFP reporter in embryonic neural tube and absence of ectopic expression. This method involves less work to achieve increased frequencies of both transgenesis and single copy, full-length integrations. These advantages are not only relevant to rodents but also for transgenesis in all systems.

Differential expression of surface markers in mouse bone marrow mesenchymal stromal cell subpopulations with distinct lineage commitment.

Bone marrow mesenchymal stromal cells (BM MSCs) represent a heterogeneous population of progenitors with potential for generation of skeletal tissues. However the identity of BM MSC subpopulations is poorly defined mainly due to the absence of specific markers allowing in situ localization of those cells and isolation of pure cell types. Here, we aimed at characterization of surface markers in mouse BM MSCs and in their subsets with distinct differentiation potential. Using conditionally immortalized BM MSCs we performed a screening with 176 antibodies and high-throughput flow cytometry, and found 33 markers expressed in MSCs, and among them 3 were novel for MSCs and 13 have not been reported for MSCs from mice. Furthermore, we obtained clonally derived MSC subpopulations and identified bipotential progenitors capable for osteo- and adipogenic differentiation, as well as monopotential osteogenic and adipogenic clones, and thus confirmed heterogeneity of MSCs. We found that expression of CD200 was characteristic for the clones with osteogenic potential, whereas SSEA4 marked adipogenic progenitors lacking osteogenic capacity, and CD140a was expressed in adipogenic cells independently of their efficiency for osteogenesis. We confirmed our observations in cell sorting experiments and further investigated the expression of those markers during the course of differentiation. Thus, our findings provide to our knowledge the most comprehensive characterization of surface antigens expression in mouse BM MSCs to date, and suggest CD200, SSEA4 and CD140a as markers differentially expressed in distinct types of MSC progenitors.

Transposon-mediated BAC transgenesis in human ES cells.

Transgenesis is a cornerstone of molecular biology. The ability to integrate a specifically engineered piece of DNA into the genome of a living system is fundamental to our efforts to understand life and exploit its implications for medicine, nanotechnology and bioprospecting. However, transgenesis has been hampered by position effects and multi-copy integration problems, which are mainly due to the use of small, plasmid-based transgenes. Large transgenes based on native genomic regions cloned into bacterial artificial chromosomes (BACs) circumvent these problems but are prone to fragmentation. Herein, we report that contrary to widely held notions, large BAC-sized constructs do not prohibit transposition. We also report the first reliable method for BAC transgenesis in human embryonic stem cells (hESCs). The PiggyBac or Sleeping Beauty transposon inverted repeats were integrated into BAC vectors by recombineering, followed by co-lipofection with the corresponding transposase in hESCs to generate robust fluorescent protein reporter lines for OCT4, NANOG, GATA4 and PAX6. BAC transposition delivers several advantages, including increased frequencies of single-copy, full-length integration, which will be useful in all transgenic systems but especially in difficult venues like hESCs.

Targeted isolation of cloned genomic regions by recombineering for haplotype phasing and isogenic targeting.

Studying genetic variations in the human genome is important for understanding phenotypes and complex traits, including rare personal variations and their associations with disease. The interpretation of polymorphisms requires reliable methods to isolate natural genetic variations, including combinations of variations, in a format suitable for downstream analysis. Here, we describe a strategy for targeted isolation of large regions (∼35 kb) from human genomes that is also applicable to any genome of interest. The method relies on recombineering to fish out target fosmid clones from pools and thereby circumvents the laborious need to plate and screen thousands of individual clones. To optimize the method, a new highly recombineering-efficient bacterial host, including inducible TrfA for fosmid copy number amplification, was developed. Various regions were isolated from human embryonic stem cell lines and a personal genome, including highly repetitive and duplicated ones. The maternal and paternal alleles at the MECP2/IRAK 1 loci were distinguished based on identification of novel allele-specific single-nucleotide polymorphisms in regulatory regions. Additionally, we applied further recombineering to construct isogenic targeting vectors for patient-specific applications. These methods will facilitate work to understand the linkage between personal variations and disease propensity, as well as possibilities for personal genome surgery.