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During cell fate transitions, cells remodel their transcriptome, chromatin, and epigenome; however, it has been difficult to determine the temporal dynamics and cause-effect relationship between these changes at the single-cell level. Here, we employ the heterokaryon-mediated reprogramming system as a single-cell model to dissect key temporal events during early stages of pluripotency conversion using super-resolution imaging. We reveal that, following heterokaryon formation, the somatic nucleus undergoes global chromatin decompaction and removal of repressive histone modifications H3K9me3 and H3K27me3 without acquisition of active modifications H3K4me3 and H3K9ac. The pluripotency gene OCT4(POU5F1) shows nascent and mature RNA transcription within the first 24 h after cell fusion without requiring an initial open chromatin configuration at its locus. NANOG, conversely, has significant nascent RNA transcription only at 48 h after cell fusion but, strikingly, exhibits genomic reopening early on. These findings suggest that the temporal relationship between chromatin compaction and gene activation during cellular reprogramming is gene context dependent.

Cell therapy approaches hold great potential for treating retinopathies, which are currently incurable. This study addresses the problem of inadequate migration and integration of transplanted cells into the host retina. To this end, we have identified the chemokines that were most upregulated during retinal degeneration and that could chemoattract mesenchymal stem cells (MSCs). The results were observed using a pharmacological model of ganglion/amacrine cell degeneration and a genetic model of retinitis pigmentosa, from both mice and human retinae. Remarkably, MSCs overexpressing Ccr5 and Cxcr6, which are receptors bound by a subset of the identified chemokines, displayed improved migration after transplantation in the degenerating retina. They also led to enhanced rescue of cell death and to preservation of electrophysiological function. Overall, we show that chemokines released from the degenerating retinae can drive migration of transplanted stem cells, and that overexpression of chemokine receptors can improve cell therapy-based regenerative approaches.

In this study, Ritschka et al. investigated whether cellular senescence might play a role in loss of regenerative capacity during liver regeneration. They show that following partial hepatectomy, the senescence-associated genes p21, p16Ink4a, and p19Arfbecome dynamically expressed in different cell types when regenerative capacity decreases, but without a full senescent response, and that treatment with a senescence-inhibiting drug improves regeneration through targeting aberrantly prolonged p21 expression.

In rodents and humans, the liver can efficiently restore its mass after hepatectomy. This is largely attributed to the proliferation and cell cycle re-entry of hepatocytes. On the other hand, bone marrow cells (BMCs) migrate into the liver after resection. Here, we find that a block of BMC recruitment into the liver severely impairs its regeneration after the surgery. Mobilized hematopoietic stem and progenitor cells (HSPCs) in the resected liver can fuse with hepatocytes, and the hybrids proliferate earlier than the hepatocytes. Genetic ablation of the hybrids severely impairs hepatocyte proliferation and liver mass regeneration. Mathematical modeling reveals a key role of bone marrow (BM)-derived hybrids to drive proliferation in the regeneration process, and predicts regeneration efficiency in experimentally non-testable conditions. In conclusion, BM-derived hybrids are essential to trigger efficient liver regeneration after hepatectomy.

Over the last decades, technological breakthroughs in super-resolution microscopy have allowed us to reach molecular resolution and design experiments of unprecedented complexity. Investigating how chromatin is folded in 3-D, from the nucleosome level up to the entire genome, is becoming possible by “magic” (iMAging GenomIC), i.e. the combination of imaging and genomic approaches. This offers endless opportunities to delve into the relationship between genome structure and function. Here, we review recently achieved objectives, and the conceptual and technical challenges the field of genome architecture is currently undertaking. We discuss what we have learned so far and where we are heading. We elucidate how the different super-resolution microscopy approaches and more specifically live cell imaging have contributed to the understanding of genome folding. Moreover, we discuss how future technical developments could address remaining open questions.

The Wnt/β-catenin pathway and Nanog are key regulators of embryonic stem cell (ESC) pluripotency and the reprogramming of somatic cells. Here, we demonstrate that the repression of Dkk1 by Nanog, which leads indirectly to β-catenin activation, is essential for reprogramming after fusion of ESCs overexpressing Nanog. In addition, β-catenin is necessary in Nanog-dependent conversion of preinduced pluripotent stem cells (pre-iPSCs) into iPSCs. The activation of β-catenin by Nanog causes fluctuations of β-catenin in ESCs cultured in serum plus leukemia inhibitory factor (serum+LIF) medium, in which protein levels of key pluripotency factors are heterogeneous. In 2i+LIF medium, which favors propagation of ESCs in a ground state of pluripotency with many pluripotency genes losing mosaic expression, we show Nanog-independent β-catenin fluctuations. Overall, we demonstrate Nanog and β-catenin cooperation in establishing naive pluripotency during the reprogramming process and their correlated heterogeneity in ESCs primed toward differentiation.

Here, we describe three complementary microscopy-based approaches to quantify morphological changes of chromatin organization in cultured adherent cells: the analysis of the coefficient of variation of DNA, the measurement of DNA-free nuclear areas, and the quantification of chromatin-associated proteins at the nuclear edge. These approaches rely on confocal imaging and stochastic optical reconstruction microscopy and allow a fast and robust quantification of chromatin compaction. These approaches circumvent inter-variability between imaging conditions and apply to every type of adherent cells.

Cell-fusion-mediated somatic-cell reprogramming can be induced in culture; however, whether this process occurs in mammalian tissues remains enigmatic. Here, we show that upon activation of Wnt/β-catenin signaling, mouse retinal neurons can be transiently reprogrammed in vivo back to a precursor stage. This occurs after their spontaneous fusion with transplanted hematopoietic stem and progenitor cells (HSPCs). Moreover, we demonstrate that retinal damage is essential for cell-hybrid formation in vivo. Newly formed hybrids can proliferate, commit to differentiation toward a neuroectodermal lineage, and finally develop into terminally differentiated neurons. This results in partial regeneration of the damaged retinal tissue, with functional rescue. Following retinal damage and induction of Wnt/β-catenin signaling, cell-fusion-mediated reprogramming also occurs after endogenous recruitment of bone-marrow-derived cells in the eyes. Our data demonstrate that in vivo reprogramming of terminally differentiated retinal neurons after their fusion with HSPCs is a potential mechanism for tissue regeneration.

Wnt/β-catenin canonical pathway has very important roles both in the regulation of self-renewal and lineage differentiation of pluripotent stem cells, as well as in the reprogramming of somatic cells. In the absence of Wnt stimulation, the target genes of the pathway are repressed or down-regulated. Conversely, when β-catenin is stabilized there is an accumulation of cytoplasmic and nuclear β-catenin that allows up-regulation of its target genes. However, the molecular mechanisms downstream of β-catenin that regulate self-renewal of embryonic stem cells remain unclear. It has been proposed that β-catenin acts through members of the T-cell factor (TCF) family. Recent studies have confirmed the role of Tcf3 (Tcf7l1) as a transcriptional repressor in embryonic stem cells, and they have also proposed a new mechanistic view as to how activation of the Wnt/β-catenin pathway can overcome this repression.

Cell‐cell fusion contributes to cell differentiation and developmental processes. We have previously showed that activation of Wnt/β‐catenin enhances somatic cell reprograming after polyethylene glycol (PEG)‐mediated fusion. Here, we show that neural stem cells and ESCs can fuse spontaneously in cocultures, although with very low efficiency (about 2%), as the hybrids undergo apoptosis. In contrast, when Wnt/β‐catenin signaling is activated in ESCs and leads to accumulation of low amounts of β‐catenin in the nucleus, activated ESCs can reprogram somatic cells with very high efficiency after spontaneous fusion. Furthermore, we also show that different levels of β‐catenin accumulation in the ESC nuclei can modulate cell proliferation, although in our experimental setting, cell proliferation does not modulate the reprograming efficiency per se. Overall, the present study provides evidence that spontaneous fusion occurs, while the survival of the reprogramed clones is strictly dependent on induction of a Wnt‐mediated reprograming pathway. STEM CELLS 2010;28:1940–1949

In all cells of the body, differentiation from zygotes has been considered a unidirectional route. Recently, however, it has become clear that the reverse path is indeed possible, in the reprogramming of somatic nuclei, i.e. the de-differentiation of somatic cells into pluripotent stem-like cells. Breakthrough discoveries have shown that over-expression of a combination of transcription factors can allow a somatic cell nucleus to be reprogrammed. Furthermore, very recently it has been shown that modulation of the Wnt/β-catenin, MAPK/ERK and PI3K/Akt signalling pathways strikingly enhances reprogramming efficiency. Despite these important findings, the molecular mechanisms driving reprogramming are still largely unknown, and moreover, it is not clear if and how these different routes for the triggering of somatic cell reprogramming converge, i.e. the over-expression of transcription factors and the modulation of signalling pathways. Here, we consider these different reprogramming routes and discuss how they can cooperate in parallel to enhance somatic cell reprogramming.

ABSTRACTThe RETproto-oncogene encodes a tyrosine kinase receptor expressed in neuroectoderm-derived cells. Mutations in specific regions of the gene are responsible for the tumor syndromes multiple endocrine neoplasia types 2A and 2B (MEN 2A and 2B), while mutations along the entire gene are involved in a developmental disorder of the gastrointestinal tract, Hirschsprung’s disease (HSCR disease). Two mutants in the extracellular domain of RET, one associated with HSCR disease and one carrying a flag epitope, were analyzed to investigate the impact of the mutations on RET function. Both mutants were impeded in their maturation, resulting in the lack of the 170-kDa mature form and the accumulation of the 150-kDa immature form in the endoplasmic reticulum. Although not exposed on the cell surface, the 150-kDa species formed dimers and aggregates; this was more pronounced in a double mutant bearing a MEN 2A mutation. Tyrosine phosphorylation and the transactivation potential were drastically reduced in single and double mutants. Finally, in cotransfection experiments both mutants exerted a dominant negative effect over protoRETand RET2A through the formation of a heteromeric complex that prevents their maturation and function. These results suggest that HSCR mutations in the extracellular region cause RET loss of function through a dominant negative mechanism.

We demonstrate that a Hirschsprung (HSCR) mutation in the tyrosine kinase domain of the RET proto-oncogene abolishes in cis the tyrosine-phosphorylation associated with the activating mutation in multiple endocrine neoplasia type 2A (MEN2A) in transiently transfected Cos cells. Yet the double mutant RET2AHS retains the ability to form stable dimers, thus dissociating the dimerization from the phosphorylation potential. Co-transfection experiments with single and double mutants carrying plasmids RET2A and RET2AHS in different ratios drastically reduced the phosphorylation levels of the RET2A protein, suggesting a dominant-negative effect of the HSCR mutation. Also, the phosphorylation associated with the multiple endocrine neoplasia type 2B (MEN2B) allele was affected in experiments with single and double mutants carrying plasmids co-transfected under the same conditions. Finally, analysis of the enzymic activity of MEN2A and MEN2B tumours confirmed the relative levels of tyrosine phosphorylation observed in Cos cells, indicating that this condition, in vivo, may account for the RET transforming potential.

Nucleosomes form heterogeneous groups in vivo, named clutches. Clutches are smaller and less dense in mouse embryonic stem cells (ESCs) compared to neural progenitor cells (NPCs). Using coarse-grained modeling of the pluripotency Pou5f1gene, we show that the genome-wide clutch differences between ESCs and NPCs can be reproduced at a single gene locus. Larger clutch formation in NPCs is associated with changes in the compaction and internucleosome contact probability of the Pou5f1fiber. Using single-molecule tracking (SMT), we further show that the core histone protein H2B is dynamic, and its local mobility relates to the structural features of the chromatin fiber. H2B is less stable and explores larger areas in ESCs compared to NPCs. The amount of linker histone H1 critically affects local H2B dynamics. Our results have important implications for how nucleosome organization and H2B dynamics contribute to regulate gene activity and cell identity.

Bi-species, fusion-mediated, somatic cell reprogramming allows precise, organism-specific tracking of unknown lineage drivers. The fusion of Tcf7l1−/−murine embryonic stem cells with EBV-transformed human B cell lymphocytes, leads to the generation of bi-species heterokaryons. Human mRNA transcript profiling at multiple time points permits the tracking of the reprogramming of B cell nuclei to a multipotent state. Interrogation of a human B cell regulatory network with gene expression signatures identifies 8 candidate master regulator proteins. Of these 8 candidates, ectopic expression of BAZ2B,from the bromodomain family, efficiently reprograms hematopoietic committed progenitors into a multipotent state and significantly enhances their long-term clonogenicity, stemness, and engraftment in immunocompromised mice. Unbiased systems biology approaches let us identify the early driving events of human B cell reprogramming.