Pluripotent Differentiation Potential Research Articles

Mesenchymal stem cells derived from hard palate: Anattractive alternative for regenerative medicine.

The palatal mucosa exhibits a notable ability to regenerate without causing scarring during the process of wound healing, rendering it a highly valuable reservoir of mesenchymal stem cells (MSCs). The aim of this review is to summarize the different sources of MSCs derived from hard palatal (PMSCs), thereby presenting a promising avenue for the utilization of regenerative medicine. Pertinent literatures focused on the sources, identification methods, and advantageous characteristics of PMSCs are obtained from PubMed and Web of Science. PMSCs, originating from the hard palate periosteum, subepithelial adipose tissue, and lamina propria, have been successfully isolated and characterized, with positive markers for MSCs and negative markers for hematopoietic stem cells. Moreover, PMSCs demonstrate resistance to inflammatory stimuli, enabling uninterrupted osteogenesis in the presence of inflammation. Additionally, PMSCs possess a notable migratory capacity, facilitating prompt arrival at the site of injury. Furthermore, PMSCs exhibit various advantageous inherent in stem cells, including clonogenicity, self-renewal capability, and pluripotent differentiation potential. PMSCs have stem cell-related properties and can be used for regenerative medicine of cells and tissues in the future.

Pilot Study for Isolation of Stromal Vascular Fraction with Collagenase Using an Automated Processing System.

There are many potential therapeutic applications for autologous adipose-derived stromal cells. These cells are found in a heterogeneous population isolated from adipose tissue called the stromal vascular fraction (SVF). Closed automated systems are available to release cells from the adherent stroma. Here, we test one system to evaluate the heterogeneous output for yield, purity, cellular characterization, and stemness criteria. The SVF was isolated from three donors using the Automated Cell Station (ACS) from BSL Co., Ltd., Busan, Republic of Korea. The SVF cellular output was characterized for cell yield and viability, immunophenotyping analysis, pluripotent differentiation potential, adhesion to plastic, and colony-forming units. Additionally, the SVF was tested for endotoxin and collagenase residuals. The SVF yield from the ACS system was an average volume of 7.9 ± 0.5 mL containing an average of 19 × 106 nucleated cells with 85 ± 12% viability. Flow cytometry identified a variety of cells, including ASCs (23%), macrophages (24%), endothelial cells (5%), pericytes (4%), and transitional cells (0.5%). The final concentrated product contained cells capable of differentiating into adipogenic, chondrogenic, and osteogenic phenotypes. Furthermore, tests for SVF sterility and purity showed no evidence of endotoxin or collagenase residuals. The ACS system can efficiently process cells from adipose tissue within the timeframe of a single surgical procedure. The cellular characterization indicated that this system can yield a sterile and concentrated SVF output, providing a valuable source of ASCs within the heterogeneous cell population.

Selecting Monoclonal Cell Lineages from Somatic Reprogramming Using Robotic-Based Spatial-Restricting Structured Flow.

Somatic cell reprogramming generates induced pluripotent stem cells (iPSCs), which serve as a crucial source of seed cells for personalized disease modeling and treatment in regenerative medicine. However, the process of reprogramming often causes substantial lineage manipulations, thereby increasing cellular heterogeneity. As a consequence, the process of harvesting monoclonal iPSCs is labor-intensive and leads to decreased reproducibility. Here, we report the first in-house developed robotic platform that uses a pin-tip-based micro-structure to manipulate radial shear flow for automated monoclonal iPSC colony selection (~1 s) in a non-invasive and label-free manner, which includes tasks for somatic cell reprogramming culturing, medium changes; time-lapse-based high-content imaging; and iPSCs monoclonal colony detection, selection, and expansion. Throughput-wise, this automated robotic system can perform approximately 24 somatic cell reprogramming tasks within 50 days in parallel via a scheduling program. Moreover, thanks to a dual flow-based iPSC selection process, the purity of iPSCs was enhanced, while simultaneously eliminating the need for single-cell subcloning. These iPSCs generated via the dual processing robotic approach demonstrated a purity 3.7 times greater than that of the conventional manual methods. In addition, the automatically produced human iPSCs exhibited typical pluripotent transcriptional profiles, differentiation potential, and karyotypes. In conclusion, this robotic method could offer a promising solution for the automated isolation or purification of lineage-specific cells derived from iPSCs, thereby accelerating the development of personalized medicines.

Hydrogels provide microenvironments to mesenchymal stem cells for craniofacial bone regeneration: Review.

The anatomical and physiological architecture of the craniofacial bone is intricate. Hence, the exact management of osteogenesis is necessary for the regeneration of the deficiencies that present in this area. Stem-based tissue engineering approaches, as opposed to conventional surgical intervention, induce bone growth with minimal postoperative risk and expense. Mesenchymal stem/stromal cells (MSC)'s pluripotent differentiation potential, anti-inflammatory and immunomodulatory properties underpin its versatility as a therapeutic agent in bone tissues. Inspired by the native stem cell niche, hydrogels are preferred choices to mediate cells and adapt to 3-D environment because of their outstanding swelling capabilities and similarity to natural extracellular matrices (ECMs). Due to their remarkable biocompatibility and capacity for stimulating bone regeneration, bone regeneration hydrogels have also received a great deal of interest. This review explores the opportunities of MSC based regenerative skeletal therapies, introduces the application of hydrogel scaffolds as artificial bone microenvironments for stem cells to explore its usage in craniofacial bone tissue engineering.

Targeting the epigenome of cancer stem cells in pediatric nervous system tumors.

Medulloblastoma, neuroblastoma, and pediatric glioma account for almost 30% of all cases of pediatric cancers. Recent evidence indicates that pediatric nervous system tumors originate from stem or progenitor cells and present a subpopulation of cells with highly tumorigenic and stem cell-like features. These cancer stem cells play a role in initiation, progression, and resistance to treatment of pediatric nervous system tumors. Histone modification, DNA methylation, chromatin remodeling, and microRNA regulation display a range of regulatory activities involved in cancer origin and progression, and cellular identity, especially those associated with stem cell features, such as self-renewal and pluripotent differentiation potential. Here, we review the contribution of different epigenetic mechanisms in pediatric nervous system tumor cancer stem cells. The choice between a differentiated and undifferentiated state can be modulated by alterations in the epigenome through the regulation of stemness genes such as CD133, SOX2, and BMI1 and the activation neuronal of differentiation markers, RBFOX3, GFAP, and S100B. Additionally, we highlighted the stage of development of epigenetic drugs and the clinical benefits and efficacy of epigenetic modulators in pediatric nervous system tumors.

Generation of CD16A gene knockout human embryonic stem cell line using CRISPR/Cas9

CD16A is a receptor for the Fc portion of immunoglobulin G, and is involved in the antibody dependent cellular cytotoxicity (ADCC) of nature killer cells(Zhu et al., 2020) and antibody dependent enhancement (ADE) of virus infections(Wan et al., 2020). However, the role of CD16A in human embryonic stem cell modeled development has been merely documented. Hence, to illustrate the role of CD16A in the human cell development, we reported a CD16A knockout human embryonic stem(hESC) cell line via CRISPR/Cas9 mediated gene knockout. The CD16A mutated cell line displayed normal karyotype, pluripotent stem cell marker gene expression and differentiation potential.

EndMT-derived mesenchymal stem cells: a new therapeutic target to atherosclerosis treatment.

Cardiovascular diseases, such as coronary artery disease and stroke, are the main threats to human health worldwide. Atherosclerosis, a chronic inflammatory disorder, plays a role as an initiator of all of the above-mentioned diseases. Cell therapy for diseases has attracted widespread attention. Mesenchymal stem cells (MSCs) are a type of stem cell that still exist in adults and have the characteristics of self-renewal ability, pluripotent differentiation potential, immunomodulation, tissue regeneration, anti-inflammation and low immunogenicity. In light of the properties of MSCs, some researchers have begun to target MSCs to create a possible way to alleviate atherosclerosis. Most of these studies are focused on MSC transplantation, injecting MSCs to modulate macrophages, the key inflammatory cell in atherosclerosis plaque. According to recent studies, researchers found that endothelial-to-mesenchymal transition (EndMT) has something to do with atherosclerosis development. A new cell typeMSC might also appear during the EndMT process. In this article, we summarize the characteristics of MSCs, the latest progress of MSC research and its application prospects, and in view of the process of EndMT occurring in atherosclerosis, we propose some new ideas for the treatment of atherosclerosis by targeting MSCs.

Neural stemness unifies cell tumorigenicity and pluripotent differentiation potential

Neural stemness is suggested to be the ground state of tumorigenicity and pluripotent differentiation potential. However, the relationship between these cell properties is unclear. Here, by disrupting the neural regulatory network in neural stem and cancer cells and by serial transplantation of cancer cells, we show that tumorigenicity and pluripotent differentiation potential are coupled cell properties unified by neural stemness. We show that loss of neural stemness via inhibition of SETDB1, an oncoprotein with enriched expression in embryonic neural cells during vertebrate embryogenesis, led to neuronal differentiation with reduced tumorigenicity and pluripotent differentiation potential in neural stem and cancer cells, whereas enhancement of neural stemness by SETDB1 overexpression caused the opposite effects. SETDB1 maintains a regulatory network comprising proteins involved in developmental programs and basic cellular functional machineries, including epigenetic modifications (EZH2), ribosome biogenesis (RPS3), translation initiation (EIF4G), and spliceosome assembly (SF3B1); all of these proteins are enriched in embryonic neural cells and play active roles in cancers. In addition, SETDB1 represses the transcription of genes promoting differentiation and cell cycle and growth arrest. Serial transplantation of cancer cells showed that neural stemness, tumorigenicity, and pluripotent differentiation potential were simultaneously enhanced; these effects were accompanied by increased expression of proteins involved in developmental programs and basic machineries, including SETDB1 and the abovementioned proteins, as well as by increased alternative splicing events. These results indicate that basic machineries work together to define a highly proliferative state with pluripotent differentiation potential and also suggest that neural stemness unifies tumorigenicity and differentiation potential.

Generation of a GFI1-flag knock-in human embryonic stem cell line using CRISPR-Cas9 technology

GFI1 is a DNA binding transcriptional repressor, it is shown to be an important gene associated with blood cells development and many blood diseases (Möröy et al., 2015). But the role of GFI1 in human hematopoieticdevelopment has not been known (Thambyrajah et al., 2016). To illustrate the function of GFI1 in human hematopoieticdevelopment, we constructed a GFI1-2 × flag-tag knock-in human embryonic stem cell line by CRISPR/Cas9 mediated gene targeting, and it would be the effective tool to study GFI1. The cell line could express GFI1-2 × flag-tag and can be identified with western blot and immunofluorescence. This cell line maintains stem cell morphology, and displays normal karyotype, pluripotent stem cell marker expression and differentiation potential.

Establishment of a CRISPR/Cas9-mediated GATAD2B homozygous knockout human embryonic stem cell line

As a subunit of the nucleosome remodeling and histone deacetylation (NuRD) complex, GATA zinc finger domain containing 2B (GATAD2B) plays a vital role in chromatin modification and transcriptional regulation. To further investigate the role of GATAD2B in cell fate determination of human ESCs, we generated two GATAD2B homozygous knockout human ESC lines by CRISPR/Cas9 technology. The cell line exhibits normal karyotype and typical stem cell morphology, following the high expression of pluripotent genes and differentiation potential in vitro. These cell lines will provide cell resources to investigate epigenetic regulation in extensive biological processes as menthioned above.

Single cell heterogeneity in human pluripotent stem cells

Human pluripotent stem cells (hPSCs) include human embryonic stem cells (hESCs) derived from blastocysts and human induced pluripotent stem cells (hiPSCs) generated from somatic cell reprogramming. Due to their self-renewal ability and pluripotent differentiation potential, hPSCs serve as an excellent experimental platform for human development, disease modeling, drug screening, and cell therapy. Traditionally, hPSCs were considered to form a homogenous population. However, recent advances in single cell technologies revealed a high degree of variability between individual cells within a hPSC population. Different types of heterogeneity can arise by genetic and epigenetic abnormalities associated with long-term in vitro culture and somatic cell reprogramming. These variations initially appear in a rare population of cells. However, some cancer-related variations can confer growth advantages to the affected cells and alter cellular phenotypes, which raises significant concerns in hPSC applications. In contrast, other types of heterogeneity are related to intrinsic features of hPSCs such as asynchronous cell cycle and spatial asymmetry in cell adhesion. A growing body of evidence suggests that hPSCs exploit the intrinsic heterogeneity to produce multiple lineages during differentiation. This idea offers a new concept of pluripotency with single cell heterogeneity as an integral element. Collectively, single cell heterogeneity is Janus-faced in hPSC function and application. Harmful heterogeneity has to be minimized by improving culture conditions and screening methods. However, other heterogeneity that is integral for pluripotency can be utilized to control hPSC proliferation and differentiation.

Neural is Fundamental: Neural Stemness as the Ground State of Cell Tumorigenicity and Differentiation Potential.

Tumorigenic cells are similar to neural stem cells or embryonic neural cells in regulatory networks, tumorigenicity and pluripotent differentiation potential. By integrating the evidence from developmental biology, tumor biology and evolution, I will make a detailed discussion on the observations and propose that neural stemness underlies two coupled cell properties, tumorigenicity and pluripotent differentiation potential. Neural stemness property of tumorigenic cells can hopefully integrate different observations/concepts underlying tumorigenesis. Neural stem cells and tumorigenic cells share regulatory networks; both exhibit neural stemness, tumorigenicity and pluripotent differentiation potential; both depend on expression or activation of ancestral genes; both rely primarily on aerobic glycolytic metabolism; both can differentiate into various cells/tissues that are derived from three germ layers, leading to tumor formation resembling severely disorganized or more degenerated process of embryonic tissue differentiation; both are enriched in long genes with more splice variants that provide more plastic scaffolds for cell differentiation, etc. Neural regulatory networks, which include higher levels of basic machineries of cell physiological functions and developmental programs, work concertedly to define a basic state with fast cell cycle and proliferation. This is predestined by the evolutionary advantage of neural state, the ground or initial state for multicellularity with adaptation to an ancient environment. Tumorigenesis might represent a process of restoration of neural ground state, thereby restoring a state with fast proliferation and pluripotent differentiation potential in somatic cells. Tumorigenesis and pluripotent differentiation potential might be better understood from understanding neural stemness, and cancer therapy should benefit more from targeting neural stemness.

Retraction of: Long-Term Maintenance of Undifferentiated Human Embryonic and Induced Pluripotent Stem Cells in Suspension (doi.org/10.1089/scd.2010.0517).

Stem Cells and DevelopmentAhead of Print Retraction NoticeOpen AccessRetraction of: Long-Term Maintenance of Undifferentiated Human Embryonic and Induced Pluripotent Stem Cells in Suspension (doi.org/10.1089/scd.2010.0517)Published Online:5 Oct 2021https://doi.org/10.1089/scd.2010.0517.retractAboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Stem Cells and Development officially retracts the paper entitled, “Long-Term Maintenance of Undifferentiated Human Embryonic and Induced Pluripotent Stem Cells in Suspension,” by Mehran Rezaei Larijani, Ali Seifinejad, Behshad Pournasr, Vahid Hajihoseini, Seydeh-Nafiseh Hassani, Mehdi Totonchi, Maryam Yousefi, Farnaz Shamsi, Ghasem Hosseini Salekdeh, and Hossein Baharvand (Stem Cells Dev 2011; 20(11):1911-1923) due to concerns regarding replicated panels within Figure 3.The Editor-in-Chief of Stem Cells and Development invited the corresponding author, Dr. Hossein Baharvand, to respond to the issues raised. Dr. Baharvand's response was as follows: “The experiment related to Figure 3A has been performed more than 10 years ago in our laboratory and the paper has been published in 2011. Unfortunately, at this point, we could not retrieve the original gel images to investigate the source of this possible error. However, we stand by our statement regarding the maintained differentiation potential of these pluripotent stem cell lines under three-dimensional culture. Pluripotency was also separately confirmed with directed differentiation and expression of the markers in Figures 3B and 3C. Furthermore, the differentiation potential of these suspension-expanded human pluripotent stem cell lines has been shown in our other studies.1-7Importantly, our conclusions remain unchanged regarding human pluripotent stem cell differentiation potential into three-germ layer derivatives using the proposed method. Finally, the data in figure 3A has no bearing on the main conclusion of our manuscript, that is our approach maintained the capability of freeze/thawing, karyotype stability, transcriptome, and pluripotency of hESCs and hiPSCs when expanded in stirred bioreactors.The authors apologize to the community for the inconvenience this has caused and request that our article to be retracted to avoid any potential ambiguities in the future.”Based on this explanation, the Editor of Stem Cells and Development agrees that the article requires retraction.The Editor and Publisher of Stem Cells and Development are committed to preserving the scientific literature and the community it serves.

Generation of human induced pluripotent stem cell lines from 2 patients with MIRAGE syndrome

MIRAGE syndrome is a multisystem disorder caused by mutations in SAMD9 (sterile α motif domain-containing protein 9) with a high mortality in the first decade of life. We generated 2 human induced pluripotent stem cell lines from male children diagnosed with MIRAGE syndrome. The cell lines were generated from fibroblasts by integration-free reprogramming using the Sendai virus. Both cell lines were fully characterized regarding their pluripotent identity and differentiation potential, and quality controlled for karyotypic integrity, cell line identity and clearance of reprogramming vectors. The generated cell lines represent a valuable tool to study the disease mechanism of MIRAGE syndrome.

Decreased Colony-Forming Ability of Subacromial Bursa-Derived Cells During Revision Arthroscopic Rotator Cuff Repair

PurposeTo compare the cellular viability and differentiation potential of subacromial bursa–derived cells (SBDCs) located over the rotator cuff muscle and tendon of patients undergoing primary versus revision arthroscopic rotator cuff repair (ARCR).MethodsSubacromial bursa was harvested from 18 primary (57.1 ± 4.6 years) and 12 revision ARCRs (57.3 ± 6.7 years). Bursa was collected from 2 sites (over rotator cuff tendon and muscle), digested with collagenase, and grown in culture. The number of nucleated cells, colony-forming units (CFUs), differentiation potential, and mesenchymal stem cell surface markers were compared in primary and revision cases.ResultsThere was no difference in the number of nucleated cells between primary and revision ARCR harvested from the subacromial bursa overlying the tendon (3019.3 ± 1420.6 cells/mg and 3541.7 ± 2244.2 cells/mg, respectively; P = .912) or muscle (2753.5 ± 1547.1 cells/mg and 2989.0 ± 2231.4 cells/mg, respectively; P = .777). There was no difference in the number of CFUs between primary and revision ARCR over the rotator cuff tendon (81.5 ± 49.5 CFUs and 53.0 ± 36.9 CFUs, respectively; P = .138), but there were significantly fewer CFUs over the muscle in revision cases (28.1 ± 22.7 CFUs) compared with primary cases (55.7 ± 34.5 CFUs) (P = .031). SBDCs from revision ARCR expressed characteristic mesenchymal stem cell surface epitopes and had multidifferentiation potentials for chondrogenesis, osteogenesis, and adipogenesis.ConclusionsSBDCs harvested over the rotator cuff muscle demonstrated significantly decreased colony-forming abilities in revision arthroscopic rotator cuff repairs compared with primary repairs. However, the subacromial bursa retains its pluripotent differentiation potential for chondrogenic, osteogenic, and adipogenic lineages in the revision setting.Clinical RelevanceThe subacromial bursa may play a role in the healing response of the repaired rotator cuff. This capacity is not necessarily diminished in the revision setting and may be harnessed as an orthobiologic.

Generation of GPAM knockout human embryonic stem cell line SYSUe-008-A using CRISPR/Cas9

GPAM (glycerol-3-phosphateacyltransferase1) is a mitochondrial enzyme that catalyze an essential step in glycerolphospholipids and triacylglycerol biosynthesis process. Loss-of-function mutation of GPAM has been shown to lead to hypomyelination of corticospinal tract in cerebral palsy patient. To model this rare disease with human brain organoid, we generated a GPAM knockout human embryonic stem cell line SYSUe-008-A by CRISPR/cas9. The GPAM knockout cell line maintains a normal karyotype and shows comparable level of pluripotent stem cell marker expression and differentiation potential as wild-type human embryonic stem cells.

Generation of PARP1 gene knockout human embryonic stem cell line using CRISPR/Cas9

PARP1 encodes a chromatin-associated enzyme which responsible for post-translational poly(ADP-ribosyl)ation modification (Hsieh et al., 2017). It plays an important role in nucleotide excision repair, non-homologous end joining, DNA mismatch repair and many other DNA repair process. Also, PARP1 participates in inflammation and aging. However, its role in human embryonic stem cell biology has not been fully resolved. To clarify the function of PARP1 in human embryonic stem cells, we reported a PARP1 knockout human embryonic stem cell line, generated by CRISPR/Cas9 mediated gene targeting. This cell line shows normal karyotype, pluripotent stem cell marker expression and differentiation potential in vitro.

Single-Cell Transcriptional Heterogeneity Landscapes of Third Heart Field Progenitor Cells

The transcriptional heterogeneity of third heart field cardiac progenitor cells (THF-CPCs) and their mechanisms of transcriptional regulation are currently unknown. This study aimed to determine the single-cell transcriptional landscapes and to reveal the transcriptional heterogeneity of THF-CPCs from embryonic and postnatal mouse hearts.Subsequently, scRNA-seq and analysis were performed with the Seurat and Monocle 3 packages in R software with standard procedures. Tbx18 and Nkx2.5 mRNA expression was determined with in situ hybridization. Immunofluorescence and β-galactosidase staining were performed to analyze reporter gene expression in mouse lineage tracing studies. Single-cell transcriptomic analysis revealed that the embryonic mouse heart contains a pool of multipotent CPCs, including first heart field, second heart field and third heart field (THF) CPC lineages. Furthermore, the key marker genes Tbx18, Wt1 and Tcf21 showed overlap in THF-CPCs, and this independent progenitor population showed unique transcriptional and spatiotemporal heterogeneity in the developing heart. According to transcriptional heterogeneity, THF-CPCs can be divided into typical, atypical and fusion cell sub-clusters. According to the spatiotemporal heterogeneity, at least, there are two types, “young”THF-CPCs, and “aged”THF-CPCs; furtherly, extracellular matrix (ECM)signal are sustained expressed in“aged”THF-CPCs, this maybe the key reasons of its loss of the pluripotent differentiation potential after birth. According to gene regulatory networks,THF-CPCs include the following cell types at least: cardiomyocyte and non-cardiomyocyte epicardial epithelial cells, which also includes components of cardiac progenitor cells.Interestingly, the transcriptional signals of epicardial epithelial cells and cardiomyocytes were clearly distinct; for example, the Tcf21 and Twist1 regulons were enriched in epicardial epithelial cells. The tbx18 regulon lacked gene regulatory networks involved in the regulation of myocardial structure, function, differentiation and proliferation. Acta1 and Tnni1 were enriched in the Hif1a regulon, which is involved in the regulation of myocardial cell structure and function. Snai1 and Tcf21 were enriched in the Twist1 regulon, which is involved in the transcriptional regulation of fibroblasts (Tgfbi and Cdh11) and smooth muscle cells (Myocd).It revealed that cellular diversity and heterogeneity of THF-CPCs was regulated by various regulons; the epithelial–mesenchymal transition (EMT) related regulons may play an important role in regulating the differentiation of fibroblasts and smooth muscle cells of THF-CPCs.Tbx18, Wt1 and TCF21-expressing cells should be defined as THF-CPCs. These cells also exist in cardiomyocytes during early embryonic development. Spatial and temporal heterogeneity may be an important reason explaining the controversy regarding THF-CPCs.

Generation of PRMT6 homozygous knockout human embryonic stem cell lines.

PRMT6 is a type I protein arginine methyltransferase (PRMT) which participates in diverse biological processes. To facilitate the understanding of PRMT6 functions, we generated two independent homozygous PRMT6 knockout Human ESCs clones, PRMT6-KO-10 and PRMT6-KO-24, in human WA01 ES cells by CRISPR/Cas9. The pluripotency of both clones was verified by the presence of pluripotent markers, normal karyotypes, and differentiation potential in vivo whereas the lack of PRMT6 expression was demonstrated by sequencing and detection of the significant changes in specific histone methylation patterns.

Generation of GADD45A gene knockout human embryonic stem cell line using CRISPR/Cas9.

GADD45A is a DNA damage and stressful growth arrest inducible protein, also it is shown to a be tumor suppressor gene and a chromatin relaxer associated with opening chromatin during the somatic reprogramming. However, its role in human embryonic stem cells and human embryonic stem cell modeled development has been merely documented. To illustrate the function of GADD45A in the human embryonic stem cell biology, we reported a GADD45A knockout human embryonic stem cell line by CRISPR/Cas9 mediated gene targeting. This cell line displayed normal karyotype, pluripotent stem cell marker expression and differentiation potential both in vivo and vitro.