Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination

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基于人多潜能干细胞的人类疾病研究与治疗

人多潜能干细胞(hPSC)包括人胚胎干细胞(hESC)和诱导性多潜能干细胞(hiPSC),理论上具有分化成为人类所有细胞类型的能力.基于hPSC的基因打靶技术,不但可以纠正人基因组中的遗传突变用于细胞治疗,还可以通过反向遗传学的方式向hPSC引入疾病特异的突变.将携带人类疾病遗传基因的hPSC分化为特定的细胞类型,在理论上可以在体外模拟人类疾病的发生,研究人类疾病发生的机理,并建立体外筛选平台寻找治疗性药物.基因编辑和干细胞技术的结合将为人类疾病的机制研究和再生医学治疗带来革命性的突破.     

High Oxygen Condition Facilitates the Differentiation of Mouse and Human Pluripotent Stem Cells into Pancreatic Progenitors and Insulin-producing Cells

Pluripotent stem cells have potential applications in regenerative medicine for diabetes. Differentiation of stem cells into insulin-producing cells has been achieved using various protocols. However, both the efficiency of the method and potency of differentiated cells are insufficient. Oxygen tension, the partial pressure of oxygen, has been shown to regulate the embryonic development of several organs, including pancreatic β-cells. In this study, we tried to establish an effective method for the differentiation of induced pluripotent stem cells (iPSCs) into insulin-producing cells by culturing under high oxygen (O₂) conditions. Treatment with a high O₂ condition in the early stage of differentiation increased insulin-positive cells at the terminus of differentiation. We found that a high O₂ condition repressed Notch-dependent gene Hes1 expression and increased Ngn3 expression at the stage of pancreatic progenitors. This effect was caused by inhibition of hypoxia-inducible factor-1α protein level. Moreover, a high O₂ condition activated Wnt signaling. Optimal stage-specific treatment with a high O₂ condition resulted in a significant increase in insulin production in both mouse embryonic stem cells and human iPSCs and yielded populations containing up to 10% C-peptide-positive cells in human iPSCs. These results suggest that culturing in a high O₂ condition at a specific stage is useful for the efficient generation of insulin-producing cells.     

A Chemical Probe that Labels Human Pluripotent Stem Cells

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Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells

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综述:基于人多潜能干细胞的人类疾病研究与治疗

人多潜能干细胞(hPSC)包括人胚胎干细胞(hESC)和诱导性多潜能干细胞(hiPSC),理论上具有分化成为人类所有细胞类型的能力.基于hPSC的基因打靶技术,不但可以纠正人基因组中的遗传突变用于细胞治疗,还可以通过反向遗传学的方式向hPSC引入疾病特异的突变.将携带人类疾病遗传基因的hPSC分化为特定的细胞类型,在理论上可以在体外模拟人类疾病的发生,研究人类疾病发生的机理,并建立体外筛选平台寻找治疗性药物.基因编辑和干细胞技术的结合将为人类疾病的机制研究和再生医学治疗带来革命性的突破.     

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)¹. While improvements in several gene delivery methods have been described^2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor¹¹, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.     

The Frequency of Homologous Recombination in Human ALT Cells

Instead of telomerase, some immortal cells use the alternative lengthening of telomeres pathway (ALT) to maintain their telomeres. There is good evidence that homologous recombination contributes to the ALT mechanism. Using an inducible GFP reporter system to measure the frequency of homologous recombination, we asked whether or not ALT cells exhibited a general change of the recombination machinery. Our results show that the frequency of homologous recombination for non-telomeric sequences in ALT cells is identical to that in telomerase positive cells, irrespective of whether the reporter was present at an intra-chromosomal location or next to a telomeric sequence. We conclude that the underlying recombination defect in ALT cells is restricted to telomeric sequences.     

A Roadmap for Human Liver Differentiation from Pluripotent Stem Cells

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Human Induced Pluripotent Stem Cells on Autologous Feeders

Background

For therapeutic usage of induced Pluripotent Stem (iPS) cells, to accomplish xeno-free culture is critical. Previous reports have shown that human embryonic stem (ES) cells can be maintained in feeder-free condition. However, absence of feeder cells can be a hostile environment for pluripotent cells and often results in karyotype abnormalities. Instead of animal feeders, human fibroblasts can be used as feeder cells of human ES cells. However, one still has to be concerned about the existence of unidentified pathogens, such as viruses and prions in these non-autologous feeders.

Methodology/Principal Findings

This report demonstrates that human induced Pluripotent Stem (iPS) cells can be established and maintained on isogenic parental feeder cells. We tested four independent human skin fibroblasts for the potential to maintain self-renewal of iPS cells. All the fibroblasts tested, as well as their conditioned medium, were capable of maintaining the undifferentiated state and normal karyotypes of iPS cells. Furthermore, human iPS cells can be generated on isogenic parental fibroblasts as feeders. These iPS cells carried on proliferation over 19 passages with undifferentiated morphologies. They expressed undifferentiated pluripotent cell markers, and could differentiate into all three germ layers via embryoid body and teratoma formation.

Conclusions/Significance

These results suggest that autologous fibroblasts can be not only a source for iPS cells but also be feeder layers. Our results provide a possibility to solve the dilemma by using isogenic fibroblasts as feeder layers of iPS cells. This is an important step toward the establishment of clinical grade iPS cells.     

Scalable Passaging of Adherent Human Pluripotent Stem Cells

Current laboratory methods used to passage adherent human pluripotent stem cells (hPSCs) are labor intensive, result in reduced cell viability and are incompatible with larger scale production necessary for many clinical applications. To meet the current demand for hPSCs, we have developed a new non-enzymatic passaging method using sodium citrate. Sodium citrate, formulated as a hypertonic solution, gently and efficiently detaches adherent cultures of hPSCs as small multicellular aggregates with minimal manual intervention. These multicellular aggregates are easily and reproducibly recovered in calcium-containing medium, retain a high post-detachment cell viability of 97%±1% and readily attach to fresh substrates. Together, this significantly reduces the time required to expand hPSCs as high quality adherent cultures. Cells subcultured for 25 passages using this novel sodium citrate passaging solution exhibit characteristic hPSC morphology, high levels (>80%) of pluripotency markers OCT4, SSEA-4, TRA-1-60 andTRA-1-81, a normal G-banded karyotype and the ability to differentiate into cells representing all three germ layers, both in vivo and in vitro.     

Homologous Recombination DNA Repair Genes Play a Critical Role in Reprogramming to a Pluripotent State

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Highlights? Reprogramming increases the level of γH2AX, a marker of DNA DSBs ? Homologous recombination (HR) deficiency impairs reprogramming ? Reprogramming-induced DSBs and HR phenotypes are method-independent ? p53 deletion rescues reprogramming defects in HR-deficient cells     

Variations in Glycogen Synthesis in Human Pluripotent Stem Cells with Altered Pluripotent States

Human pluripotent stem cells (hPSCs) represent very promising resources for cell-based regenerative medicine. It is essential to determine the biological implications of some fundamental physiological processes (such as glycogen metabolism) in these stem cells. In this report, we employ electron, immunofluorescence microscopy, and biochemical methods to study glycogen synthesis in hPSCs. Our results indicate that there is a high level of glycogen synthesis (0.28 to 0.62 μg/μg proteins) in undifferentiated human embryonic stem cells (hESCs) compared with the glycogen levels (0 to 0.25 μg/μg proteins) reported in human cancer cell lines. Moreover, we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway. Our observation of glycogen bodies and sustained expression of the pluripotent factor Oct-4 mediated by the potent GSK-3 inhibitor CHIR-99021 reveals an altered pluripotent state in hPSC culture. We further confirmed glycogen variations under different naïve pluripotent cell growth conditions based on the addition of the GSK-3 inhibitor BIO. Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states, similar to those naïve-like hPSCs, with increased glycogen synthesis. Furthermore, we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis. Thus, our novel findings regarding the dynamic changes in glycogen metabolism provide new markers to assess the energetic and various pluripotent states in hPSCs. The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions.     

Targeting Homologous Recombination in Notch-Driven C. elegans Stem Cell and Human Tumors

Mammalian NOTCH1-4 receptors are all associated with human malignancy, although exact roles remain enigmatic. Here we employ glp-1(ar202), a temperature-sensitive gain-of-function C. elegans NOTCH mutant, to delineate NOTCH-driven tumor responses to radiotherapy. At ≤20°C, glp-1(ar202) is wild-type, whereas at 25°C it forms a germline stem cell⁄progenitor cell tumor reminiscent of human cancer. We identify a NOTCH tumor phenotype in which all tumor cells traffic rapidly to G2⁄M post-irradiation, attempt to repair DNA strand breaks exclusively via homology-driven repair, and when this fails die by mitotic death. Homology-driven repair inactivation is dramatically radiosensitizing. We show that these concepts translate directly to human cancer models.     

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.