Eriona Hysolli

Neuronal maturation defect in induced pluripotent stem cells from patients with Rett syndrome

Rett syndrome (RTT) is one of the most prevalent female neurodevelopmental disorders that cause severe mental retardation. Mutations in methyl CpG binding protein 2 (MeCP2) are mainly responsible for RTT. Patients with classical RTT exhibit normal development until age 6–18 mo, at which point they become symptomatic and display loss of language and motor skills, purposeful hand movements, and normal head growth. Murine genetic models and postmortem human brains have been used to study the disease and enable the molecular dissection of RTT. In this work, we applied a recently developed reprogramming approach to generate a novel in vitro human RTT model. Induced pluripotent stem cells (iPSCs) were derived from RTT fibroblasts by overexpressing the reprogramming factors OCT4, SOX2, KLF4, and MYC. Intriguingly, whereas some iPSCs maintained X chromosome inactivation, in others the X chromosome was reactivated. Thus, iPSCs were isolated that retained a single active X chromosome expressing either mutant or WT MeCP2, as well as iPSCs with reactivated X chromosomes expressing both mutant and WT MeCP2. When these cells underwent neuronal differentiation, the mutant monoallelic or biallelelic RTT-iPSCs displayed a defect in neuronal maturation consistent with RTT phenotypes. Our in vitro model of RTT is an important tool allowing the further investigation of the pathophysiology of RTT and the development of the curative therapeutics.

Human induced pluripotent stem cells and neurodegenerative disease: prospects for novel therapies.

PURPOSE OF REVIEW The lack of effective treatments for various neurodegenerative disorders has placed huge burdens on society. We review the current status in applying induced pluripotent stem cell (iPSC) technology for the cellular therapy, drug screening, and in-vitro modeling of neurodegenerative diseases. RECENT FINDINGS iPSCs are generated from somatic cells by overexpressing four reprogramming factors (Oct4, Sox2, Klf4, and Myc). Like human embryonic stem cells, iPSCs have features of self-renewal and pluripotency, and allow in-vitro disease modeling, drug screening, and cell replacement therapy. Disease-specific iPSCs were derived from patients of several neurodegenerative diseases, including Parkinsons disease, Alzheimers disease, amyotrophic lateral sclerosis, and spinal muscular atrophy. Neurons differentiated from these iPSCs recapitulated the in-vivo phenotypes, providing platforms for drug screening. In the case of Parkinsons disease, iPSC-derived dopaminergic neurons gave positive therapeutic effect on a rodent Parkinsons disease model as a proof of principle in using iPSCs as sources of cell replacement therapy. Beyond iPSC technology, much effort is being made to generate neurons directly from dermal fibroblasts with neuron-specific transcription factors, which does not require making iPSCs as an intermediate cell type. SUMMARY We summarize recent progress in using iPSCs for modeling the progress and treatment of neurodegenerative diseases and provide evidence for future perspectives in this field.

X Chromosome of Female Cells Shows Dynamic Changes in Status during Human Somatic Cell Reprogramming

Summary Induced pluripotent stem cells (iPSCs) acquire embryonic stem cell (ESC)-like epigenetic states, including the X chromosome. Previous studies reported that human iPSCs retain the inactive X chromosome of parental cells, or acquire two active X chromosomes through reprogramming. Most studies investigated the X chromosome states in established human iPSC clones after completion of reprogramming. Thus, it is still not fully understood when and how the X chromosome reactivation occurs during reprogramming. Here, we report a dynamic change in the X chromosome state throughout reprogramming, with an initial robust reactivation of the inactive X chromosome followed by an inactivation upon generation of nascent iPSC clones. iPSCs with two active X chromosomes or an eroded X chromosome arise in passaging iPSCs. These data provide important insights into the plasticity of the X chromosome of human female iPSCs and will be crucial for the future application of such cells in cell therapy and X-linked disease modeling.

Regulation of the DNA Methylation Landscape in Human Somatic Cell Reprogramming by the miR-29 Family

Summary Reprogramming to pluripotency after overexpression of OCT4, SOX2, KLF4, and MYC is accompanied by global genomic and epigenomic changes. Histone modification and DNA methylation states in induced pluripotent stem cells (iPSCs) have been shown to be highly similar to embryonic stem cells (ESCs). However, epigenetic differences still exist between iPSCs and ESCs. In particular, aberrant DNA methylation states found in iPSCs are a major concern when using iPSCs in a clinical setting. Thus, it is critical to find factors that regulate DNA methylation states in reprogramming. Here, we found that the miR-29 family is an important epigenetic regulator during human somatic cell reprogramming. Our global DNA methylation and hydroxymethylation analysis shows that DNA demethylation is a major event mediated by miR-29a depletion during early reprogramming, and that iPSCs derived from miR-29a depletion are epigenetically closer to ESCs. Our findings uncover an important miRNA-based approach to generate clinically robust iPSCs.

Transcriptome Signature and Regulation in Human Somatic Cell Reprogramming

Summary Reprogramming of somatic cells produces induced pluripotent stem cells (iPSCs) that are invaluable resources for biomedical research. Here, we extended the previous transcriptome studies by performing RNA-seq on cells defined by a combination of multiple cellular surface markers. We found that transcriptome changes during early reprogramming occur independently from the opening of closed chromatin by OCT4, SOX2, KLF4, and MYC (OSKM). Furthermore, our data identify multiple spliced forms of genes uniquely expressed at each progressive stage of reprogramming. In particular, we found a pluripotency-specific spliced form of CCNE1 that is specific to human and significantly enhances reprogramming. In addition, single nucleotide polymorphism (SNP) expression analysis reveals that monoallelic gene expression is induced in the intermediate stages of reprogramming, while biallelic expression is recovered upon completion of reprogramming. Our transcriptome data provide unique opportunities in understanding human iPSC reprogramming.

Transformation of somatic cells into stem cell-like cells under a stromal niche

To study the genomic plasticity of somatic cells without ectopic genetic manipulation, we cultured mouse fibroblasts with ovarian cells, embryonic fibroblasts of different strains, and parthenogenetic embryonic stem cells (ESCs). Of 41 trials, cell aggregation resembling nascent ESC colony from inner cell mass was detected in 9 cases (22%), and 6 cases (67%) yielded fibroblast‐derived colonies with ESC morphology. Cells used in coculture provided the critical (P=0.0061) inducing factor for the aggregation. These colony‐forming fibroblasts (CFFs) showed similar characteristics to those in ESCs and induced pluripotent stem cells (iPSCs), including pluripotency gene expression, in vitro differentiation, and teratoma formation. Furthermore, CFFs produced somatic chimera, although none showed germline chimerism. CFFs had a tetraploid‐like karyotype, and their imprinting patterns differed from parthenogenetic ESCs, thereby confirming their nongermline transmissibility. We observed dysregulation of cell cycle‐related proteins, as well as both homologous and heterologous recombination of genomic single‐nucleotide polymorphisms in CFFs. Our observations provide information on somatic cell plasticity, resulting in stemness or tumorigenesis, regardless of colony‐forming cell progenitors in the fibroblast population. The plasticity of somatic genomes under environmental influences, as well as acquisition of pluripotency by cell fusion, is also implicated.—Lee, S. T., Gong, S. P., Yum, K. E., Lee, E. J., Lee, C. H., Choi, J. H., Kim, D. Y., Han, H., Kim, K.‐S., Hysolli, E., Ahn, J. Y., Park, I.‐H., Han, J. Y., Jeong, J.‐W., Lim, J. M. Transformation of somatic cells into stem cell‐like cells under a stromal niche. FASEB J. 27, 2644‐2656 (2013). www.fasebj.org

The lesser known story of X-chromosome reactivation: A closer look into the reprogramming of the inactive X chromosome

X-chromosome inactivation (XCI) is an important mechanism employed by mammalian XX female cells to level X-linked gene expression with that of male XY cells. XCI occurs early in development as the pluripotent cells of the inner cell mass (ICM) in blastocysts successively differentiate into cells of all three germ layers. X-chromosome reactivation (XCR), the reversal of XCI, is critical for germ cell formation as a mechanism to diversify the X-chromosome gene pool. Here we review the characterization of XCR, and further explore its natural occurrence during development and the in vitro models of cellular reprogramming. We also review the key regulators involved in XCI for their role in suppressing the active histone marks and the genes in the active chromosome for their inhibition of X inactivation signals.

From Designing the Molecules of Life to Designing Life: Future Applications Derived from Advances in DNA Technologies

Since the elucidation of its structure, DNA has been at the forefront of biological research. In the past half century, an explosion of DNA-based technology development has occurred with the most rapid advances being made for DNA sequencing. In parallel, dramatic improvements have also been made in the synthesis and editing of DNA from the oligonucleotide to the genome scale. In this Review, we will summarize four different subfields relating to DNA technologies following this trajectory of smaller to larger scale. We begin by talking about building materials out of DNA which in turn can act as delivery vehicles in vivo. We then discuss how altering microbial genomes can lead to novel methods of production for industrial biologics. Next, we talk about the future of writing whole genomes as a method of studying evolution. Lastly, we highlight the ways in which barcoding biological systems will allow for their three-dimensional analysis in a highly multiplexed fashion.

Assembly of Radically Recoded E. coli Genome Segments

The large potential of radically recoded organisms (RROs) in medicine and industry depends on improved technologies for efficient assembly and testing of recoded genomes for biosafety and functionality. Here we describe a next generation platform for conjugative assembly genome engineering, termed CAGE 2.0, that enables the scarless integration of large synthetically recoded E. coli segments at isogenic and adjacent genomic loci. A stable tdk dual selective marker is employed to facilitate cyclical assembly and removal of attachment sites used for targeted segment delivery by sitespecific recombination. Bypassing the need for vector transformation harnesses the multi Mb capacity of CAGE, while minimizing artifacts associated with RecA-mediated homologous recombination. Our method expands the genome engineering toolkit for radical modification across many organisms and recombinase-mediated cassette exchange (RMCE).

Reprogramming human somatic cells into induced pluripotent stem cells (iPSCs) using retroviral vector with GFP.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine(1). It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)(2-4) . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone(4). Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.