Yixuan Wang

Genetic correction of β-thalassemia patient-specific iPS cells and its use in improving hemoglobin production in irradiated SCID mice

The generation of induced pluripotent stem cells (iPSCs) from differentiated somatic cells by over-expression of several transcription factors has the potential to cure many genetic and degenerative diseases currently recalcitrant to traditional clinical approaches. One such genetic disease is β-thalassemia major (Cooleys anemia). This disease is caused by either a point mutation or the deletion of several nucleotides in the β-globin gene, and it threatens the lives of millions of people in China. In the present study, we successfully generated iPSCs from fibroblasts collected from a 2-year-old patient who was diagnosed with a homozygous 41/42 deletion in his β-globin gene. More importantly, we successfully corrected this genetic mutation in the β-thalassemia iPSCs by homologous recombination. Furthermore, transplantation of the genetically corrected iPSCs-derived hematopoietic progenitors into sub-lethally irradiated immune deficient SCID mice showed improved hemoglobin production compared with the uncorrected iPSCs. Moreover, the generation of human β-globin could be detected in the mice transplanted with corrected iPSCs-derived hematopietic progenitors. Our study provides strong evidence that iPSCs generated from a patient with a genetic disease can be corrected by homologous recombination and that the corrected iPSCs have potential clinical uses.

RCOR2 Is a Subunit of the LSD1 Complex That Regulates ESC Property and Substitutes for SOX2 in Reprogramming Somatic Cells to Pluripotency

Histone demethylase LSD1 can form complex with different Rcor family corepressors in different cell types. It remains unknown if cell‐specific Rcor proteins function specifically in distinct cell types. Here, we report that Rcor2 is predominantly expressed in ESCs and forms a complex with LSD1 and facilitates its nucleosomal demethylation activity. Knockdown of Rcor2 in ESCs inhibited ESC proliferation and severely impaired the pluripotency. Moreover, knockdown of Rcor2 greatly impaired the formation of induced pluripotent stem (iPS) cells. In contrast, ectopic expression of Rcor2 in somatic cells together with Oct4, Sox2, and Klf4 promoted the formation of iPS cells. Most interestingly, ectopic expression of Rcor2 in both mouse and human somatic cells effectively substituted the requirement for exogenous Sox2 expression in somatic cell reprogramming. STEM CELLS 2011;

Novel Importin-α Family Member Kpna7 Is Required for Normal Fertility and Fecundity in the Mouse

Nuclear importing system and nuclear factors play important roles in mediating nuclear reprogramming and zygotic gene activation. However, the components and mechanisms that mediate nuclearly specific targeting of the nuclear proteins during nuclear reprogramming and zygotic gene activation remain largely unknown. Here, we identified a novel member of the importin-α family, AW146299(KPNA7), which is predominantly expressed in mouse oocytes and zygotes and localizes to the nucleus or spindle. Mutation of Kpna7 gene caused reproductivity reduction and sex imbalance by inducing preferential fetal lethality in females. Parthenogenesis analysis showed that the cell cycle of activated one-cell embryos is loss of control and ahead of schedule but finally failed to develop into blastocyst stage. Further RT-PCR and epigenetic modification analysis showed that knocking out of Kpna7 induced abnormalities of gene expression (dppa2, dppa4, and piwil2) and epigenetic modifications (down-regulation of histone H3K27me3). Biochemical analysis showed that KPNA7 interacts with KPNB1 (importin-β1). In summary, we identified a novel Kpna7 gene that is required for normal fertility and fecundity.

Generation of induced pluripotent stem cells from human β-thalassemia fibroblast cells

Induced pluripotent stem (iPS) cells have recently been generated by directly introducing several transcription factors into differentiated human somatic cells, and these iPS cells show great similarities to embryo-derived ES cells [1-3]. Moreover, patient-specific iPS cells have recently been generated, and these studies provided hopes for patients with genetic and degenerative diseases [4, 5]. β-thalassemia is an inherited blood disorder that is characterized by reduced synthesis of hemoglobin subunit beta (hemoglobin β-chain). Individuals with thalassemia major (also called Cooleys anemia) have severe anemia and hepatosplenomegaly; without treatment , affected children have severe failure to thrive and a shortened life expectancy. Even with transfusion and chelation therapy treatments, the life span of patients with thalassemia major can only be extended for a limited time. More importantly, β-thalassemia patients are widely distributed throughout the southern part of China, and this genetically inherited disease has threatened millions of peoples lives for decades with no effective treatment available. Here, we report that we have successfully generated β-thalassemia-specific iPS cells, which may pave the way to optimize life span-extending treatments for patients with β-thalassemia major. We used a previously published protocol [6] to generate β-thalassemia patient-specific iPS cells. Retroviruses containing human OCT3/4, SOX2, KLF4, and c-MYC were introduced into fibroblast cells derived from a ho-mozygous β-thalassemia individual. To monitor infection efficiency, a GFP-expressing plasmid pMXs-GFP was used as a control. The single-gene infection efficiency reached approximately 80%-90%, as determined by GFP expression. Six days after transduction, the cells were harvested by trypsinization and plated onto mitomycin C-treated MEF feeder cells at a density of 1×10 5 cells per 100-mm dish. Two days later, the medium was replaced with the one typically used for human ES (hES) cell culture that was supplemented with 10 ng/ml basic fibro-blast growth factor. Approximately 2 weeks after the transduced cells were plated on the feeder cells, two kinds of colonies appeared. Some were granulated colonies that were not similar in morphology to hES cells, whereas the others closely resembled hES cell colonies. These colonies exhibited a flat and tightly packed morphology with sharp edges, and had a high nucleus/cytoplasm ratio and large nucleoli. From each plate, approximately 10 to 50 hES-like colonies were obtained, and these colonies were individually isolated and mechanically passaged around 25 days after infection. These cells were passaged every 3-5 days, and they displayed morphologies and growth rates that are similar to those of hES cells …

Naïve Induced Pluripotent Stem Cells Generated From β-Thalassemia Fibroblasts Allow Efficient Gene Correction With CRISPR/Cas9

Conventional primed human embryonic stem cells and induced pluripotent stem cells (iPSCs) exhibit molecular and biological characteristics distinct from pluripotent stem cells in the naïve state. Although naïve pluripotent stem cells show much higher levels of self‐renewal ability and multidifferentiation capacity, it is unknown whether naïve iPSCs can be generated directly from patient somatic cells and will be superior to primed iPSCs. In the present study, we used an established 5i/L/FA system to directly reprogram fibroblasts of a patient with β‐thalassemia into transgene‐free naïve iPSCs with molecular signatures of ground‐state pluripotency. Furthermore, these naïve iPSCs can efficiently produce cross‐species chimeras. Importantly, using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 nuclease genome editing system, these naïve iPSCs exhibit significantly improved gene‐correction efficiencies compared with the corresponding primed iPSCs. Furthermore, human naïve iPSCs could be directly generated from noninvasively collected urinary cells, which are easily acquired and thus represent an excellent cell resource for further clinical trials. Therefore, our findings demonstrate the feasibility and superiority of using patient‐specific iPSCs in the naïve state for disease modeling, gene editing, and future clinical therapy.

LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

Epigenetic regulatory complexes play key roles in the modulation of transcriptional regulation underlying neural stem cell (NSC) proliferation and progeny specification. How specific cofactors guide histone demethylase LSD1/KDM1A complex to regulate distinct NSC-related gene activation and repression in cortical neurogenesis remains unclear. Here we demonstrate that Rcor2, a co-repressor of LSD1, is mainly expressed in the central nervous system (CNS) and plays a key role in epigenetic regulation of cortical development. Depletion of Rcor2 results in reduced NPC proliferation, neuron population, neocortex thickness and brain size. We find that Rcor2 directly targets Dlx2 and Shh, and represses their expressions in developing neocortex. In addition, inhibition of Shh signals rescues the neurogenesis defects caused by Rcor2 depletion both in vivo and in vitro. Hence, our findings suggest that co-repressor Rcor2 is critical for cortical development by repressing Shh signalling pathway in dorsal telencephalon.

Epigenetic regulation of somatic cell reprogramming

Pluripotent stem cells, having self-renewal capacities and multi-lineage differentiation abilities, offer great potential in disease modeling and therapeutic applications. The successful generation of induced pluripotent stem cells (iPSCs) by the Yamanaka group in 2006 is a milestone event in both reprogramming and stem cell research fields, which makes in vitro somatic cell reprogramming and personalized stem cell therapy feasible. During the past 10 years, several important progresses have been made in uncovering the molecular mechanisms involved in the reprogramming process, which shed light on improving the reprogramming efficiency and iPSC quality. Here, we briefly review the important progresses in the epigenetic regulation including histone and DNA modifications during somatic cell reprogramming.

Reprogramming of H3K9me3-dependent heterochromatin during mammalian embryo development

H3K9me3-dependent heterochromatin is a major barrier of cell fate changes that must be reprogrammed after fertilization. However, the molecular details of these events are lacking in early embryos. Here, we map the genome-wide distribution of H3K9me3 modifications in mouse early embryos. We find that H3K9me3 exhibits distinct dynamic features in promoters and long terminal repeats (LTRs). Both parental genomes undergo large-scale H3K9me3 reestablishment after fertilization, and the imbalance in parental H3K9me3 signals lasts until blastocyst. The rebuilding of H3K9me3 on LTRs is involved in silencing their active transcription triggered by DNA demethylation. We identify that Chaf1a is essential for the establishment of H3K9me3 on LTRs and subsequent transcriptional repression. Finally, we find that lineage-specific H3K9me3 is established in post-implantation embryos. In summary, our data demonstrate that H3K9me3-dependent heterochromatin undergoes dramatic reprogramming during early embryonic development and provide valuable resources for further exploration of the epigenetic mechanism in early embryos.Gao and colleagues characterize genome-wide H3K9me3 distributions in pre- and post-implantation mouse embryos, providing a resource to further our understanding of epigenomic dynamics during mammalian embryogenesis.

Direct induction of neural progenitor cells transiently passes through a partially reprogrammed state

The generation of functional neural progenitor cells (NPCs) holds great promise for both research and clinical applications in neurodegenerative diseases. Traditionally, NPCs are derived from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), or NPCs can be directly converted from somatic cells by sets of transcription factors or by a combination of chemical cocktails and/or hypoxia. However, the ethical issues of ESCs, the risk of tumorigenesis from iPSCs and transgenic integration from exogenous genes as well as complicated manipulation and time-consuming of chemical induced NPCs (ciNPCs) limit the applications of these strategies. Here, we describe a novel method for generating growth factor-induced neural progenitor cells (giNPCs) from mouse embryonic and adult fibroblasts by using inductive and/or permissive signaling culture conditions. These giNPCs closely resemble brain-derived NPCs in terms of transcription networks and neural lineage differentiation potentials. Moreover, this somatic cell to NPC induction is a gradual process that includes initiation, intermediate, maturation and stabilization stages. Importantly, gene expression and histone modification analyses further indicate a partially reprogrammed state during the generation process of induced NPCs, in which lineage specific genes and pluripotency associated genes are transiently activated. Our study therefore describes the potential safety problems that also exist in the transgene-free direct induction strategy and highlights the importance of excluding the possibility of residual partially reprogrammed and/or teratoma-like cells from the generated NPCs for future clinical trials.

Unique molecular events during reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) at naïve state

Derivation of human naïve cells in the ground state of pluripotency provides promising avenues for developmental biology studies and therapeutic manipulations. However, the molecular mechanisms involved in the establishment and maintenance of human naïve pluripotency remain poorly understood. Using the human inducible reprogramming system together with the 5iLAF naïve induction strategy, integrative analysis of transcriptional and epigenetic dynamics across the transition from human fibroblasts to naïve iPSCs revealed ordered waves of gene network activation sharing signatures with those found during embryonic development from late embryogenesis to pre-implantation stages. More importantly, Transcriptional analysis showed a significant transient reactivation of transcripts with 8-cell-stage-like characteristics in the late stage of reprogramming, suggesting transient activation of gene network with human zygotic genome activation (ZGA)-like signatures during the establishment of naïve pluripotency. Together, Dissecting the naïve reprogramming dynamics by integrative analysis improves the understanding of the molecular features involved in the generation of naïve pluripotency directly from somatic cells.