Zhili Rong

Immunogenicity of induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells with defined factors, hold great promise for regenerative medicine as the renewable source of autologous cells. Whereas it has been generally assumed that these autologous cells should be immune-tolerated by the recipient from whom the iPSCs are derived, their immunogenicity has not been vigorously examined. We show here that, whereas embryonic stem cells (ESCs) derived from inbred C57BL/6 (B6) mice can efficiently form teratomas in B6 mice without any evident immune rejection, the allogeneic ESCs from 129/SvJ mice fail to form teratomas in B6 mice due to rapid rejection by recipients. B6 mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs by either retroviral approach (ViPSCs) or a novel episomal approach (EiPSCs) that causes no genomic integration. In contrast to B6 ESCs, teratomas formed by B6 ViPSCs were mostly immune-rejected by B6 recipients. In addition, the majority of teratomas formed by B6 EiPSCs were immunogenic in B6 mice with T cell infiltration, and apparent tissue damage and regression were observed in a small fraction of teratomas. Global gene expression analysis of teratomas formed by B6 ESCs and EiPSCs revealed a number of genes frequently overexpressed in teratomas derived from EiPSCs, and several such gene products were shown to contribute directly to the immunogenicity of the B6 EiPSC-derived cells in B6 mice. These findings indicate that, in contrast to derivatives of ESCs, abnormal gene expression in some cells differentiated from iPSCs can induce T-cell-dependent immune response in syngeneic recipients. Therefore, the immunogenicity of therapeutically valuable cells derived from patient-specific iPSCs should be evaluated before any clinic application of these autologous cells into the patients.

An effective approach to prevent immune rejection of human ESC-derived allografts.

Human embryonic stem cells (hESCs) hold great promise for cell therapy as a source of diverse differentiated cell types. One key bottleneck to realizing such potential is allogenic immune rejection of hESC-derived cells by recipients. Here, we optimized humanized mice (Hu-mice) reconstituted with a functional human immune system that mounts a vigorous rejection of hESCs and their derivatives. We established knockin hESCs that constitutively express CTLA4-Ig and PD-L1 before and after differentiation, denoted CP hESCs. We then demonstrated that allogenic CP hESC-derived teratomas, fibroblasts, and cardiomyocytes are immune protected in Hu-mice, while cells derived from parental hESCs are effectively rejected. Expression of both CTLA4-Ig, which disrupts T cell costimulatory pathways, and PD-L1, which activates T cell inhibitory pathway, is required to confer immune protection, as neither was sufficient on their own. These findings are instrumental for developing a strategy to protect hESC-derived cells from allogenic immune responses without requiring systemic immune suppression.

hSef inhibits PC-12 cell differentiation by interfering with ras-mitogen-activated protein kinase MAPK signaling

Growth factor signaling by receptor tyrosine kinases regulates several cell fates, such as proliferation and differentiation. Sef was genetically identified as a negative regulator of fibroblast growth factor (FGF) signaling. Using bioinformatic methods and rapid amplification of cDNA ends-PCR, we isolated both the mouse and the human Sef genes, which encoded the Sef protein and Sef-S isoform that was generated through alternative splicing. We provide evidence that the Sef gene products were located mainly on the cell membrane. Co-immunoprecipitation and immunostaining experiments indicate that hSef interacts with FGFR1 and FGFR2 but not FGFR3. Our results demonstrated that stably expressed hSef strongly inhibits FGF2- or nerve growth factor-induced PC-12 cell differentiation. The intracellular domain of hSef is necessary for the inhibitory effect on FGF2-induced PC-12 cell differentiation. Furthermore, our data suggested Sef exerted the negative effect on FGF2-induced PC-12 cell differentiation through the prevention of Ras-mitogen-activated protein kinase signaling, possibly functioning upstream of the Ras molecule. These findings suggest that Sef may play an important role in the regulation of PC-12 cell differentiation.

IL-17RD (Sef or IL-17RLM) interacts with IL-17 receptor and mediates IL-17 signaling

Interleukin-17 (IL-17 or IL-17A) production is a hallmark of TH17 cells, a new unique lineage of CD4+ T lymphocytes contributing to the pathogenesis of multiple autoimmune and inflammatory diseases. IL-17 receptor (IL-17R or IL-17RA) is essential for IL-17 biological activity. Emerging data suggest that the formation of a heteromeric and/or homomeric receptor complex is required for IL-17 signaling. Here we show that the orphan receptor IL-17RD (Sef, similar expression to FGF genes or IL-17RLM) is associated and colocalized with IL-17R. Importantly, IL-17RD mediates IL-17 signaling, as evaluated using a luciferase reporter driven by the native promoter of 24p3, an IL-17 target gene. In addition, an IL-17RD mutant lacking the intracellular domain dominant-negatively suppresses IL-17R-mediated IL-17 signaling. Moreover, IL-17RD as well as IL-17R is associated with TRAF6, an IL-17R downstream molecule. These results indicate that IL-17RD is a part of the IL-17 receptor signaling complex, therefore providing novel evidence for IL-17 signaling through a heteromeric and/or homomeric receptor complex.

A scalable approach to prevent teratoma formation of human embryonic stem cells

Background: One of the major hurdles hindering the clinic development of hESC-based therapy is the teratoma risk. Results: A genetic modification via homologous recombination can effectively eliminate hESCs without killing their derivatives. Conclusion: A scalable and safe approach to eliminate the teratoma risk associated with hESCs. Significance: Our approach improves the feasibility to develop hESCs into human cell therapy. As the renewable source of all cell types in the body, human embryonic stem cells (hESCs) hold great promise for human cell therapy. However, one major bottleneck that hinders the clinic application of hESCs is that hESCs remaining with their differentiated derivatives pose cancer risk by forming teratomas after transplantation. NANOG is a critical pluripotency factor specifically expressed in hESCs but rarely in their differentiated derivatives. By introducing a hyperactive variant of herpes simplex virus thymidine kinase gene into the 3′-untranslated region of the endogenous NANOG gene of hESCs through homologous recombination, we developed a safe and highly scalable approach to efficiently eliminate the teratoma risk associated with hESCs without apparent negative impact on their differentiated cell types. As thymidine kinase is widely used in human gene therapy trials and is the therapeutic target of U. S. Food and Drug Administration-approved drugs, our strategy could be effectively applied to the clinic development of hESC-based human cell therapy.

siRNA targeting the Leader sequence of SARS-CoV inhibits virus replication

SARS-CoV (the SARS-Associated Coronavirus) was reported as a novel virus member in the coronavirus family, which was the cause of severe acute respiratory syndrome. Coronavirus replication occurs through a unique mechanism employing Leader sequence in the transcripts when initiating transcription from the genome. Therefore, we cloned the Leader sequence from SARS-CoV(BJ01), which is identical to that identified from SARS-CoV(HKU-39849), and constructed specific siRNA targeting the Leader sequence. Using EGFP and RFP reporter genes fused with the cloned SARS-CoV Leader sequence, we demonstrated that the siRNA targeting the Leader sequence decreased the mRNA abundance and protein expression levels of the reporter genes in 293T cells. By stably expressing the siRNA in Vero E6 cells, we provided data that the siRNA could effectively and specifically decrease the mRNA abundance of SARS-CoV genes as analyzed by RT-PCR and Northern blot. Our data indicated that the siRNA targeting the Leader sequence inhibited the replication of SARS-CoV in Vero E6 cells by silencing gene expression. We further demonstrated, via transient transfection experiments, that the siRNA targeting the Leader sequence had a much stronger inhibitory effect on SARS-CoV replication than the siRNAs targeting the Spike gene or the antisense oligodeoxynucleotides did. This report provides evidence that targeting Leader sequence using siRNA could be a powerful tool in inhibiting SARS-CoV replication.

Transmembrane helix of novel oncogene with kinase-domain (NOK) influences its oligomerization and limits the activation of RAS/MAPK signaling

Ligand-dependent or independent oligomerization of receptor protein tyrosine kinase (RPTK) is often an essential step for receptor activation and intracellular signaling. The novel oncogene with kinase-domain (NOK) is a unique RPTK that almost completely lacks an ectodomain, expresses intracellularly and activates constitutively. However, it is unknown whether NOK can form oligomer or what function oligomerization would have. In this study, two NOK deletion mutants were generated by either removing the ectodomain (NOKΔECD) or including the endodomain (NOK-ICD). Co-immunoprecipitation demonstrated that the transmembrane (TM) domain of NOK was essential for its intermolecular interaction. The results further showed that NOK aggregated more closely as lower order oligomers (the dimer- and trimer-sized) than either deletion mutant did since NOK could be cross-linked by both Sulfo-EGS and formaldehyde, whereas either deletion mutant was only sensitive to Sulfo-EGS. Removing the NOK TM domain (NOK-ICD) not only markedly promoted higher order oligomerization, but also altered the subcellular localization of NOK and dramatically elevated the NOK-mediated constitutive activation of extracellular signal-regulated kinase (ERK). Moreover, NOK-ICD but not NOK or NOKΔECD was co-localized with the upstream signaling molecule RAS on cell membrane. Thus, TM-mediated intermolecular contacting may be mainly responsible for the constitutive activation of NOK and contribute to the autoinhibitory effect on RAS/MAPK signaling.

A Safety Checkpoint to Eliminate Cancer Risk of the Immune Evasive Cells Derived from Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) hold great promise in the regenerative therapy of many currently untreatable human diseases. One of the key bottlenecks is the immune rejection of hESC‐derived allografts by the recipient. To overcome this challenge, we have established new approaches to induce immune protection of hESC‐derived allografts through the coexpression of immune suppressive molecules CTLA4‐Ig and PD‐L1. However, this in turn raises a safety concern of cancer risk because these hESC‐derived cells can evade immune surveillance. To address this safety concern, we developed a safety checkpoint so that the immune evasive hESC‐derived cells in the graft can be effectively eliminated if any cellular transformation is detected. In this context, we knock‐in the suicidal gene herpes simplex virus thymidine kinase (HSVTK) into the constitutive HPRT locus of CP hESCs (knock‐in hESCs expressing CTLA4‐Ig and PD‐L1), denoted CPTK hESCs. Employing humanized mice (Hu‐mice) reconstituted with human immune system, we demonstrated that the CPTK hESC‐derived cells are protected from immune rejection. In addition, CPTK hESC‐derived cells can be efficiently eliminated in vitro and in vivo with FDA approved TK‐targeting drug ganciclovir. Therefore, this new safety checkpoint improves the feasibility to use the immune evasive hESC‐derived cells for regenerative medicine. Stem Cells 2017;35:1154–1161

Gene Targeting Through Homologous Recombination in Monkey Embryonic Stem Cells Using CRISPR/Cas9 System.

Editor: Genome editing is a critical tool in both basic biomedical research and gene therapy. Compared with zinc finger nucleases and transcription activator-like effectors, the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system offers a more efficient and user-friendly design and utilizes DNA-RNA recognition instead of DNA-protein recognition, and it has been successfully applied in various species [1]. The nonhuman primate is ideal to model human diseases and evaluate the potential applications of embryonic stem cells (ESCs) in regenerative medicine. Therefore, it will be important to develop a technology to genetically manipulate monkey ESCs. A recent report has indicated that the CRISPR/Cas9 system has recently been used to generate the gene-modified cynomolgus monkey [2]. While this is an important proof-of-concept experiment, a genetically modified nonhuman primate is expensive to maintain and requires facility that is only available to a small number of laboratories. Instead, monkey ESCs can undergo unlimited self-renewal while maintaining the potential to give rise to all cell types and therefore an in vitro alternative of the monkey model. In this study, we demonstrated the feasibility to genetically modify the ESCs of rhesus monkey using the CRISPR/Cas9 system for cell tracing. To test whether spCas9 can induce precise gene targeting by means of homologous recombination (HR) in rhesus monkey ESCs, we designed the experiment to employ spCas9 and a long DNA donor template to introduce the Brainbow2.1 coding region into the HPRT locus, which is a housing keeping gene on X-chromosome [3] (Fig. 1A). We used the Brainbow gene that can be used for cell tracing [4]. In this context, the Cre-loxP recombination can be used to create a stochastic choice of XFP expression, resulting in the heritable marking of ESC-derived cells with multiple distinct colors. In the long DNA donor template (Fig. 1B), a Brainbow2.1 expression cassette (CAG-Brainbow2.1-polyA) and a selection cassette (CAG-neo-IRES-Puro-polyA) were inserted into the two HPRT homologous arms around 1 kb (Fig. 1C). Four sgRNAs (Fig. 1D), which targeted the junction site of the two homologous arms, were designed according to the (N)20NGG rule (See Supplementary Data for sgRNA; Supplementary materials are available online at www.liebertpub.com/scd). It was expected to make double nicking of genomic DNA by paired sgRNAs (sgRNA3 and sgRNA4) and the mutant Cas9 (Cas9n) that confers a higher targeting specificity and is applicable in human ESCs and induced pluripotent stem cells [5,6]. After electroporation and puromycin selection, 80 monkey ESC clones survived by means of cotransfection of four single sgRNAs with wt Cas9 and paired sgRNAs (sgRNA3 and sgRNA4) with Cas9n. We used PCR screening to identify 22 positive clones (See Supplementary Data for Primer sequences) (Fig. 1E). To further confirm HR, we used Southern blotting with the AvrII digestion and hybridization to the upstream, downstream, and internal probes (See Supplementary Data for Probe sequences). As shown in Fig. 1F, 9 of the 22 clones had precise HR at both arms with no random integration. The knockin ESC lines (termed ormBB cells) exhibited typical ESC morphology (Fig. 1J-a). To determine the pluripotency of ormBB cells, we performed teratoma assays. When injected into severe combined immune deficiency (SCID) mice, both ormES22 (WT) and ormBB cells formed well-differentiated teratomas, and subsequent histological analysis identified representative cell types of all three germ layers (Fig. 1H). To test the expression of Brainbow fluorescent genes that can be used for single-cell labeling, the plasmid-encoding Cre recombinase was transiently electroporated into ormBB ESCs (electroporated cells were termed ormBB-Cre cells). Three days later, XFP signals were demonstrated by FACS and microscopy analyses (Fig. 1I, J-b). FIG. 1. Brainbow Knockin strategy in rhesus monkey embryonic stem cells through CRISPR/Cas9 and XFP expressions in ormBB-derived cells. (A) The endogenous rhesus monkey HPRT locus. The arrow indicates the HPRT gene. The locations of upstream and downstream probes ... To test whether ormBB cells could be applied to track cell lineage in vitro, ormBB-Cre cells were differentiated into the embryoid body followed by conditions for neural lineage differentiation [7]. XFP-expressing cells could be differentiated into typical neural rosette-like structures (Fig. 1J-c), indicating that ormBB-Cre ESCs could be used to individually labeled cells during differentiation. To test whether the cells derived from ormBB-Cre ESCs could be tracked in vivo, we took advantage of the capability of ESCs to form teratomas in SCID mice, which consisted of various cell types of the three germ layers. We harvested fresh teratomas formed by ormBB-Cre ESCs, which were cut into tiny pieces and examined under the immunofluorescence microscope. As shown in Fig. 1K, cells individually labeled by one of the four fluorescent colors could be found throughout the teratomas. Therefore, ormBB cells could be applied in individual cell tracing. To our knowledge, our report represents the first description of the genomic editing of monkey ESCs by the CRISPR/Cas9 system. In the context of WT Cas9 system, the targeting efficiency is high (33% with sgRNA3, Fig. 1G) in rhesus monkey ESCs. However, in the context of the nickase Cas9n together with paired sgRNAs (sgRNA3 and sgRNA4), the targeting efficiency is very low in monkey ESCs, consistent with the notion that Cas9n confers a much lower targeting efficiency than WT Cas9. In addition, our findings also show a high frequency of random integrations, indicating that the CRISPR/Cas9 system could target nonspecific genomic sites.

Live cell imaging of genomic loci using dCas9-SunTag system and a bright fluorescent protein

CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems have been harnessed for kinds of genome manipulation, including gene editing, transcription regulation, and chromosome loci imaging (Dominguez et al., 2016; Komor et al., 2017). A typical engineered CRISPR-Cas9 system is composed of a Cas9 protein and a single guide RNA (sgRNA), which could form a protein/RNA complex to recognize and cleave DNA sequence (Hsu et al., 2014; Wright et al., 2016). Based on nuclease-deactivated Cas9, termed dCas9, the CRISPRCas9 system can be used as an imaging tool to label genomic loci and visualize dynamic changes of chromosomes (Chen et al., 2013; Cheng et al., 2016; Fu et al., 2016; Ma et al., 2016; Qin et al., 2017; Wang et al., 2016). An EGFP-tagged dCas9 bound with a structurally optimized sgRNA enables visualization of repetitive genomic sequences in live cells (Chen et al., 2013). A CRISPRainbow system could simultaneously image up to six genomic loci in a single cell (Ma et al., 2016). However, these approaches have an obvious disadvantage, the fluorescent signal is relative weak and the signal-to-noise ratio is low, and thus the application to low or no repeats DNA sequences is limited. SunTag system is a protein-tagging system for signal amplification, consisting of an array of repeating peptide and an antibody-fusion protein, which can bind to each other (Tanenbaum et al., 2014). mNeonGreen, a monomeric yellow-green fluorescent protein derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum, is reported to be the brightest monomeric green or yellow fluorescent protein so far described (Shaner et al., 2013). In comparison experiments, mNeonGreen showed excellent properties that were superior to the most commonly used green and yellow fluorescent proteins, and thus held great potential for imaging. Considering their unique properties, we expected that application of SunTag system and mNeonGreen to CRISPR/ Cas9-based imaging might increase signal intensity, as well as signal-to-noise ratio. Therefore, 24 copies of GCN4 peptide were fused to the C-terminus of dCas9 (dCas9SunTag), and GCN4 peptide binding single-chain variable fragment antibody (scFv-GCN4) was fused to superfolderGFP (sfGFP), mNeonGreen, or three-tandem-repeats of mNeonGreen (3XmNeonGreen). As sgRNA structure could significantly affect imaging quality, a sgRNA scaffold optimized by A-U base pair flip and hairpin extension was used to eliminate protein aggregation and increase bright dot number (Chen et al., 2013). The schematic of the imaging strategy was shown in Fig. 1A. To test the hypothesis, HEK293T cells were co-transfected with dCas9-SunTag, a telomere-targeted sgRNA, and sfGFP, mNeonGreen, or 3XmNeonGreen, respectively. As shown in Fig. 1B and Movies S1, S2 and S3, all the fluorescent proteins formed puncta in cell nuclei. In a quantitative assay, about 26% cells contained 61–80 foci and about 5% cells contained 81–100 foci in sfGFP transfected cells, and about 27% cells contained 61–80 foci and about 24% cells contained 81–100 foci in mNeonGreen transfected cells, while about 38% cells contained 61–80 foci and about 10% cells contained 81–100 foci in 3XmNeonGreen transfected cells. Since the expected telomere number is about 92 at G1 cell cycle stage in a diploid human cell, the mNeonGreen strategy is the optimal one to visualize most telomere loci in a cell, compared with the other two strategies (Fig. 1C). To further assess the specificity and efficiency of telomere labeling by the three strategies, fluorescence in situ hybridization (FISH) assay using a Cy5-tagged telomere-specific-probe and immunofluorescence staining assay using a primary antibody against HA tag (each fluorescence protein contains a HA tag) and an Alexa488-labeled secondary antibody were performed in the same cells (Schmitt et al., 2010). The results showed that FISH and sfGFP, mNeonGreen, and 3XmNeonGreen signals were perfectly matched, indicating that the foci of sfGFP, mNeonGreen, and 3XmNeonGreen were indeed corresponding to telomeres and that the efficiency is similar between CRISPR imaging and FISH assay (Fig. 1D). As shown in Figure 1B and 1D, the fluorescence signal intensity and signal-to-noise ratio were obviously different, and thus a quantitative assay was performed. The fluorescence intensity of 3XmNeonGreen was higher than