ZBTB7A regulates primed-to-naïve transition of pluripotent stem cells via recognition of the PNT-associated sequence by zinc fingers 1-2 and recognition of γ-globin -200 gene element by zinc fingers 1-4.

ZBTB7A, a transcription factor containing a tandem array of four Cys2-His2 zinc fingers (ZFs), is vital for multiple physiological events through directional binding to different genomic loci. Our previously determined crystal structure of ZBTB7A in complex with a GCCCCTTCCCC sequence revealed that all four ZFs (ZF1-4) are involved in binding to γ-globin -200 gene element to repress fetal haemoglobin expression. Recently, it has been reported that ZBTB7A drives primed-to-naïve transition (PNT) of pluripotent stem cells through binding to a 12-bp consensus sequence ([AAGGACCCAGAT], referred to as PNT-associated sequence). Here, we report a crystal structure of ZBTB7A ZF1-3 in complex with the PNT-associated sequence. The structure shows that ZF1 and ZF2 primarily contribute to recognizing the GACCC core sequence mimicking the half part (GCCCC) of γ-globin -200 gene element via specific hydrogen bonding and van der Waals contacts. The mutations of key residues in ZF1-2 remarkably reduce their binding affinities for the PNT-associated sequence in vitro and cannot restore epiblast stem cells to the naïve pluripotent state in vivo. Collectively, our studies demonstrate that ZBTB7A mainly employs its ZF1-2 to recognize the PNT-associated sequence but recognizes γ-globin -200 gene element via ZF1-4, providing insights into the molecular mechanism for the diversity of ZBTB7A's genomic localization.

Combinational quorum sensing devices for dynamic control in cross-feeding cocultivation.

Quorum sensing (QS) offers cell density dependent dynamic regulations in cell culture through devices such as synchronized lysis circuit (SLC) and metabolic toggle switch (MTS). However, there is still a lack of studies on cocultivation with a combination of different QS-based devices. Taking the production of isopropanol and salidroside as case studies, we have mathematically modeled a comprehensive set of QS-regulated cocultivation schemes and constructed experimental combinations of QS devices, respectively, to evaluate their feasibility and optimality for regulating growth competition and corporative production. Glucose split ratio is proposed for the analysis of competition between cell growth and targeted production. Results show that the combination of different QS devices across multiple members offers a new tool with the potential to effectively coordinate synthetic microbial consortia for achieving high product titer in cross-feeding cocultivation. It is also evident that the performance of such systems is significantly affected by dynamic characteristics of chosen QS devices, carbon source control and the operational settings. This study offers insights for future applications of combinational QS devices in synthetic microbial consortia.

Generation of induced pluripotent stem cell GZLSL-i001-A derived from urine-derived cells of Hemophilia A patient with Inv22 mutation.

Hemophilia A (HA), is a X-linked recessive congenital bleeding disorder, caused by deficiency of the coagulation factorVIII (FVIII) which is encoded by coagulation factor 8 (F8). HA affects 1 of every 5,000 males worldwide. The intron 22 inversion (Inv22) mutation of F8 causes about 45% of severeHA cases.Here, we generated induced pluripotent stem cells (iPSCs) from a HA patient with Inv22 mutation by electroporation of urine-derived cells (UCs) with episomal plasmids under feeder-free, virus-free, serum-free condition and without oncogene c-MYC. This iPSCs line could facilitate future applications of human iPSCs by provide a valuable cell model.

Efficient gene correction of an aberrant splice site in ß-thalassaemia iPSCs by CRISPR/Cas9 and single-strand oligodeoxynucleotides.

ß-thalassaemia is a prevalent hereditary haematological disease caused by mutations in the human haemoglobin ß (HBB) gene. Among them, the HBB IVS2-654 (C > T) mutation, which is in the intron, creates an aberrant splicing site. Bone marrow transplantation for curing ß-thalassaemia is limited due to the lack of matched donors. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), as a widely used tool for gene editing, is able to target specific sequence and create double-strand break (DSB), which can be combined with the single-stranded oligodeoxynucleotide (ssODN) to correct mutations. In this study, according to two different strategies, the HBB IVS2-654 mutation was seamlessly corrected in iPSCs by CRISPR/Cas9 system and ssODN. To reduce the occurrence of secondary cleavage, a more efficient strategy was adopted. The corrected iPSCs kept pluripotency and genome stability. Moreover, they could differentiate normally. Through CRISPR/Cas9 system and ssODN, our study provides improved strategies for gene correction of ß-Thalassaemia, and the expression of the HBB gene can be restored, which can be used for gene therapy in the future.

Establishment of an ectodermal dysplasia related gene EDA Knockout human embryonic stem cell line (WAe001-A-22) by CRISPR-Cas9 technology.

EDA is a gene located at Xq13.1. It encodes different isoforms of tumor necrosis factor (TNF) superfamily member ectodysplasin A. Ectodysplasin A is a transmembrane protein which can be cleaved to form a secreted form and interact with EDA receptor to mediate the development of ectoderm. Mutations of the EDA gene are related to ectodermal dysplasia and tooth agenesis. Here, we report the establishment of the EDA gene knockout human embryonic stem (hES) cell line by CRISPR-Cas9 technology. This cell line provides good materials for further studies of the roles ectodysplasin A plays in ectoderm differentiation and tooth development.

Establishment of a congenital tooth agenesis related gene MSX1 knockout human embryonic stem cell lines by CRISPR-Cas9 technology.

Human MSX1 gene is mapped to chromosome 4 and encodes a 303aa homeobox protein MSX1. MSX1 expression appears during early tooth development of vertebrate embryogenesis. Mutations in this protein are related to human tooth anomalie, cleft lip and palate and congenital ectodermal dysplasia syndrome. Most of the confirmed pathogenic mutations are located in exon2 encoded homeobox domain. Here, we report the establishment of MSX1 gene knockout human embryonic stem (hES) cell lines by CRISPR-Cas9 technology. These cell lines provide good materials for further studies of the roles MSX1 plays in human tooth development and congenital tooth agenesis.

Generation of integration-free induced pluripotent stem cells (GZHMUi001-A) by reprogramming peripheral blood mononuclear cells from a 47, XXX syndrome patient.

47, XXX syndrome is one of several sex-chromosomal aneuploidies, and it has an incidence of approximately 1/1000 in newborn females. Because of heterogeneity in X-inactivation, these patients may exhibit a variety of clinical symptoms. Here, we report the generation of an integration-free human induced pluripotent stem cell line (GZHMUi001-A) by using Sendai virus to reprogram peripheral blood mononuclear cells from a 47, XXX syndrome patient with premature ovarian failure. This 47, XXX iPS cell line has characteristics of pluripotent stem cells and is a useful tool for the investigation of this X chromosome aneuploid disease.

Transcription activator-like effector nuclease (TALEN)-mediated gene correction in integration-free ß-thalassemia induced pluripotent stem cells.

ß-Thalassemia (ß-Thal) is a group of life-threatening blood disorders caused by either point mutations or deletions of nucleotides in ß-globin gene (HBB). It is estimated that 4.5% of the population in the world carry ß-Thal mutants (1), posing a persistent threat to public health. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations offer an ideal therapeutic solution to this problem. However, homologous recombination-based gene correction in human iPSCs remains largely inefficient. Here, we describe a robust process combining efficient generation of integration-free ß-Thal iPSCs from the cells of patients and transcription activator-like effector nuclease (TALEN)-based universal correction of HBB mutations in situ. We generated integration-free and gene-corrected iPSC lines from two patients carrying different types of homozygous mutations and showed that these iPSCs are pluripotent and have normal karyotype. We showed that the correction process did not generate TALEN-induced off targeting mutations by sequencing. More importantly, the gene-corrected ß-Thal iPS cell lines from each patient can be induced to differentiate into hematopoietic progenitor cells and then further to erythroblasts expressing normal ß-globin. Our studies provide an efficient and universal strategy to correct different types of ß-globin mutations in ß-Thal iPSCs for disease modeling and applications.

Generating a non-integrating human induced pluripotent stem cell bank from urine-derived cells.

Induced pluripotent stem cell (iPS cell) holds great potential for applications in regenerative medicine, drug discovery, and disease modeling. We describe here a practical method to generate human iPS cells from urine-derived cells (UCs) under feeder-free, virus-free, serum-free condition and without oncogene c-MYC. We showed that this approach could be applied in a large population with different genetic backgrounds. UCs are easily accessible and exhibit high reprogramming efficiency, offering advantages over other cell types used for the purpose of iPS generation. Using the approach described in this study, we have generated 93 iPS cell lines from 20 donors with diverse genetic backgrounds. The non-viral iPS cell bank with these cell lines provides a valuable resource for iPS cells research, facilitating future applications of human iPS cells.

Generation of integration-free neural progenitor cells from cells in human urine.

Human neural stem cells hold great promise for research and therapy in neural disease. We describe the generation of integration-free and expandable human neural progenitor cells (NPCs). We combined an episomal system to deliver reprogramming factors with a chemically defined culture medium to reprogram epithelial-like cells from human urine into NPCs (hUiNPCs). These transgene-free hUiNPCs can self-renew and can differentiate into multiple functional neuronal subtypes and glial cells in vitro. Although functional in vivo analysis is still needed, we report that the cells survive and differentiate upon transplant into newborn rat brain.

MicroRNA cluster 302-367 enhances somatic cell reprogramming by accelerating a mesenchymal-to-epithelial transition.

MicroRNAs (miRNAs) are emerging critical regulators of cell function that frequently reside in clusters throughout the genome. They influence a myriad of cell functions, including the generation of induced pluripotent stem cells, also termed reprogramming. Here, we have successfully delivered entire miRNA clusters into reprogramming fibroblasts using retroviral vectors. This strategy avoids caveats associated with transient transfection of chemically synthesized miRNA mimics. Overexpression of 2 miRNA clusters, 106a-363 and in particular 302-367, allowed potent increases in induced pluripotent stem cell generation efficiency in mouse fibroblasts using 3 exogenous factors (Sox2, Klf4, and Oct4). Pathway analysis highlighted potential relevant effectors, including mesenchymal-to-epithelial transition, cell cycle, and epigenetic regulators. Further study showed that miRNA cluster 302-367 targeted TGFß receptor 2, promoted increased E-cadherin expression, and accelerated mesenchymal-to-epithelial changes necessary for colony formation. Our work thus provides an interesting alternative for improving reprogramming using miRNAs and adds new evidence for the emerging relationship between pluripotency and the epithelial phenotype.