Yonglun Luo

Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9

Taking the PERVs out of pigs With the severe shortage of organs needed for transplants, xenotransplantation (transplantation of nonhuman organs to humans) offers an alternative source. Some pig organs have similar size and function to those of humans. The challenge is that the pig genome harbors porcine endogenous retroviruses (PERVs) that can potentially pass to humans with possibly damaging consequences. Niu et al. generated pigs in which all copies of PERVs were inactivated by CRISPR-Cas9 genome engineering (see the Perspective by Denner). Not only does this work provide insights into PERV activity, but it also opens the door to a safer source of organs and tissues for pig-to-human xenotransplantation. Science, this issue p. 1303; see also p. 1238 Cloned pigs can be reared that lack all porcine endogenous retroviruses. Xenotransplantation is a promising strategy to alleviate the shortage of organs for human transplantation. In addition to the concerns about pig-to-human immunological compatibility, the risk of cross-species transmission of porcine endogenous retroviruses (PERVs) has impeded the clinical application of this approach. We previously demonstrated the feasibility of inactivating PERV activity in an immortalized pig cell line. We now confirm that PERVs infect human cells, and we observe the horizontal transfer of PERVs among human cells. Using CRISPR-Cas9, we inactivated all of the PERVs in a porcine primary cell line and generated PERV-inactivated pigs via somatic cell nuclear transfer. Our study highlights the value of PERV inactivation to prevent cross-species viral transmission and demonstrates the successful production of PERV-inactivated animals to address the safety concern in clinical xenotransplantation.

Genetically modified pigs for biomedical research

During the last two decades, pigs have been used to develop some of the most important large animal models for biomedical research. Advances in pig genome research, genetic modification (GM) of primary pig cells and pig cloning by nuclear transfer, have facilitated the generation of GM pigs for xenotransplantation and various human diseases. This review summarizes the key technologies used for generating GM pigs, including pronuclear microinjection, sperm-mediated gene transfer, somatic cell nuclear transfer by traditional cloning, and somatic cell nuclear transfer by handmade cloning. Broadly used genetic engineering tools for porcine cells are also discussed. We also summarize the GM pig models that have been generated for xenotransplantation and human disease processes, including neurodegenerative diseases, cardiovascular diseases, eye diseases, bone diseases, cancers and epidermal skin diseases, diabetes mellitus, cystic fibrosis, and inherited metabolic diseases. Thus, this review provides an overview of the progress in GM pig research over the last two decades and perspectives for future development.

A simple method for deriving functional MSCs and applied for osteogenesis in 3D scaffolds

We describe a simple method for bone engineering using biodegradable scaffolds with mesenchymal stem cells derived from human induced-pluripotent stem cells (hiPS-MSCs). The hiPS-MSCs expressed mesenchymal markers (CD90, CD73, and CD105), possessed multipotency characterized by tri-lineages differentiation: osteogenic, adipogenic, and chondrogenic, and lost pluripotency – as seen with the loss of markers OCT3/4 and TRA-1-81 – and tumorigenicity. However, these iPS-MSCs are still positive for marker NANOG. We further explored the osteogenic potential of the hiPS-MSCs in synthetic polymer polycaprolactone (PCL) scaffolds or PCL scaffolds functionalized with natural polymer hyaluronan and ceramic TCP (PHT) both in vitro and in vivo. Our results showed that these iPS-MSCs are functionally compatible with the two 3D scaffolds tested and formed typically calcified structure in the scaffolds. Overall, our results suggest the iPS-MSCs derived by this simple method retain fully osteogenic function and provide a new solution towards personalized orthopedic therapy in the future.

Genetically Modified Pig Models for Neurodegenerative Disorders

Increasing incidence of neurodegenerative disorders such as Alzheimers disease and Parkinsons disease has become one of the most challenging health issues in ageing humans. One approach to combat this is to generate genetically modified animal models of neurodegenerative disorders for studying pathogenesis, prognosis, diagnosis, treatment, and prevention. Owing to the genetic, anatomic, physiologic, pathologic, and neurologic similarities between pigs and humans, genetically modified pig models of neurodegenerative disorders have been attractive large animal models to bridge the gap of preclinical investigations between rodents and humans. In this review, we provide a neuroanatomical overview in pigs and summarize and discuss the generation of genetically modified pig models of neurodegenerative disorders including Alzheimers diseases, Huntingtons disease, Parkinsons disease, amyotrophic lateral sclerosis, spinal muscular atrophy, and ataxia–telangiectasia. We also highlight how non‐invasive bioimaging technologies such as positron emission tomography (PET), computer tomography (CT), and magnetic resonance imaging (MRI), and behavioural testing have been applied to characterize neurodegenerative pig models. We further propose a multiplex genome editing and preterm recloning (MAP) approach by using the rapid growth of the ground‐breaking precision genome editing technology CRISPR/Cas9 and somatic cell nuclear transfer (SCNT). With this approach, we hope to shorten the temporal requirement in generating multiple transgenic pigs, increase the survival rate of founder pigs, and generate genetically modified pigs that will more closely resemble the disease‐causing mutations and recapitulate pathological features of human conditions. Copyright

Mesenchymal stem cells derived from human induced pluripotent stem cells retain adequate osteogenicity and chondrogenicity but less adipogenicity.

IntroductionPreviously, we established a simple method for deriving mesenchymal stem cells (MSCs) from human induced pluripotent stem cells (iPSC-MSCs). These iPSC-MSCs were capable of forming osteogenic structures in scaffolds and nanofibers. The objective of this study is to systematically characterize the mesenchymal characteristics of the iPSC-MSCs by comparing them to bone marrow-derived MSCs (BM-MSCs).MethodsTwo iPSC-MSC lines (named as mRNA-iPSC-MSC-YL001 and lenti-iPSC-MSC-A001) and one BM-MSC line were used for the study. Cell proliferation, presence of mesenchymal surface markers, tri-lineage differentiation capability (osteogenesis, chondrogenesis, adipogenesis), and expression of “stemness” genes were analyzed in these MSC lines.ResultsThe iPSC-MSCs were similar to BM-MSCs in terms of cell morphology (fibroblast-like) and surface antigen profile: CD29+, CD44+, CD73+, CD90+, CD105+, CD11b–, CD14–, CD31–, CD34–, CD45– and HLA-DR–. A faster proliferative capability was seen in both iPSC-MSCs lines compared to the BM-MSCs. The iPSC-MSCs showed adequate capacity of osteogenesis and chondrogenesis compared to the BM-MSCs, while less adipogenic potential was found in the iPSC-MSCs. The iPSC-MSCs and the tri-lineage differentiated cells (osteoblasts, chondrocytes, adipocytes) all lack expression of “stemness” genes: OCT4, SOX2, GDF3, CRIPTO, UTF1, DPPA4, DNMT3B, LIN28a, and SAL4.ConclusionsThe MSCs derived from human iPSCs with our method have advanced proliferation capability and adequate osteogenic and chondrogenic properties compared to BM-MSCs. However, the iPSC-MSCs were less efficient in their adipogenicity, suggesting that further modifications should be applied to our method to derive iPSC-MSCs more closely resembling the naïve BM-MSCs if necessary.

Gene expression responses to FUS, EWS, and TAF15 reduction and stress granule sequestration analyses identifies FET-protein non-redundant functions.

The FET family of proteins is composed of FUS/TLS, EWS/EWSR1, and TAF15 and possesses RNA- and DNA-binding capacities. The FET-proteins are involved in transcriptional regulation and RNA processing, and FET-gene deregulation is associated with development of cancer and protein granule formations in amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and trinucleotide repeat expansion diseases. We here describe a comparative characterization of FET-protein localization and gene regulatory functions. We show that FUS and TAF15 locate to cellular stress granules to a larger extend than EWS. FET-proteins have no major importance for stress granule formation and cellular stress responses, indicating that FET-protein stress granule association most likely is a downstream response to cellular stress. Gene expression analyses showed that the cellular response towards FUS and TAF15 reduction is relatively similar whereas EWS reduction resulted in a more unique response. The presented data support that FUS and TAF15 are more functionally related to each other, and that the FET-proteins have distinct functions in cellular signaling pathways which could have implications for the neurological disease pathogenesis.

Comparison of Gene Expression and Genome-Wide DNA Methylation Profiling between Phenotypically Normal Cloned Pigs and Conventionally Bred Controls

Animal breeding via Somatic Cell Nuclear Transfer (SCNT) has enormous potential in agriculture and biomedicine. However, concerns about whether SCNT animals are as healthy or epigenetically normal as conventionally bred ones are raised as the efficiency of cloning by SCNT is much lower than natural breeding or In-vitro fertilization (IVF). Thus, we have conducted a genome-wide gene expression and DNA methylation profiling between phenotypically normal cloned pigs and control pigs in two tissues (muscle and liver), using Affymetrix Porcine expression array as well as modified methylation-specific digital karyotyping (MMSDK) and Solexa sequencing technology. Typical tissue-specific differences with respect to both gene expression and DNA methylation were observed in muscle and liver from cloned as well as control pigs. Gene expression profiles were highly similar between cloned pigs and controls, though a small set of genes showed altered expression. Cloned pigs presented a more different pattern of DNA methylation in unique sequences in both tissues. Especially a small set of genomic sites had different DNA methylation status with a trend towards slightly increased methylation levels in cloned pigs. Molecular network analysis of the genes that contained such differential methylation loci revealed a significant network related to tissue development. In conclusion, our study showed that phenotypically normal cloned pigs were highly similar with normal breeding pigs in their gene expression, but moderate alteration in DNA methylation aspects still exists, especially in certain unique genomic regions.

Regulation of Peripheral Clock to Oscillation of Substance P Contributes to Circadian Inflammatory Pain

Background: The daily fluctuations of many physiologic and behavioral parameters are differentially influenced by either central or peripheral clocks in mammals. Since substance P (SP) oscillates in some brain tissues and plays an indispensable role in modulating inflammatory pain at the spinal level, we speculated that SP mediates circadian nociception transmission at the spinal level. Methods: In the present study behavioral observation, real-time polymerase chain reaction, luciferase assay, chromatin immunoprecipitation, and immunohistochemistry stain methods were used to investigate the role of SP in the spinal circadian nociception transmission and its regulation mechanism. Results: Our results showed that under transcriptional regulation of BMAL1:CLOCK heterodimers, SPs coding gene Tac1 expression oscillates in dorsal root ganglion (n = 36), but not in the spinal dorsal horn. Further, the expression of SP cycled in the spinal dorsal horn, and this rhythmicity was potentially determined by circadian expression of Tac1 in dorsal root ganglion. Furthermore, the variation of SP expression induced by formalin was fluctuated in a similar rhythm to behavioral nociceptive response induced by formalin (n = 48); and the nociceptive behavioral circadian rhythm could be abolished through blockade of the SP–Neurokinin 1 receptor pathway (n = 70). Lastly, the variations of spinal SP expression and behavioral nociceptive response were in step, and both were changed by the deletion mutation of clock gene. Conclusions: We conclude that spinal SP probably plays a pivotal role in modulating circadian inflammatory pain and suggest that peripheral circadian-regulated signaling is potentially an essential pathway for circadian nociceptive transmission.

Osteogenesis of human induced pluripotent stem cells derived mesenchymal stem cells on hydroxyapatite contained nanofibers

Biomimetic nanofibrous scaffolds combined with stem cells are promising for bone tissue engineering. In the present study, we have employed nano-hydroxyapatite (nHAp) contained polycaprolactone (PCL) nanofibers as a biomimetic nanofibrous scaffold, and mesenchymal stem cells derived from human induced pluripotent stem cells (hiPS-MSCs) as the novel stem cells sources. The response of hiPS-MSCs on the nanofibrous scaffolds in terms of cell proliferation and differentiation into the osteoblastic phenotype was investigated by XTT assay, scanning electron microscopy (SEM), osteogenic genes expression (runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), collagen I (COL1A1), and osteocalcin (OC)), ALP activity, and calcium deposition. It is clearly shown that the hiPS-MSCs attached, and proliferated on the nanofibrous scaffolds. Compared with PCL nanofibers without nHAp, the cells on the nHAp contained nanofibers demonstrated superior capabilites to differentiate to form calcified extracellular matrix. Together with gene expression, all of the results indicate the great potential of the hiPS-MSCs seeded biomimetic nanofibrous scaffolds for bone regeneration in the future.

EXPRESSION PROFILING REVEALS A POSITIVE REGULATION BY MPER2 ON CIRCADIAN RHYTHM OF CYTOTOXICITY RECEPTORS: LY49C AND NKG2D

The mammalian circadian gene, mPer2, an indispensable component of the mammalian circadian clock, not only modulates endogenous circadian rhythms but also plays a crucial role in regulating innate immune function. Previously, we showed that mPer2 plays a crucial role in regulating cytotoxic response. To investigate the molecular mechanism for mPer2-controlled cytotoxic response, in the present study we conducted mRNA expression for 11 genes participating in cytotoxicity regulation in wild-type (WT) and mPer2 knockout (mPer2  −; ; /  −; ; ) mice bone marrow, that is, Dap-10, Ly49C, Ly49I, Rac1, Mapk1, Map2k1, Nkg2d, Shp-1, Pak1, Pik3ca, and Vav1. The mRNA levels of Ly49C (p < 0.001), Ly49I (p = 0.039), and Nkg2d (p = 0.038) were significantly downregulated in mPer2  −; ; /  −; ;  mice. Time-dependence of expression profiling was then conducted for four core clock genes (Per1, Bmal1, Clock, Rev-erbα), and six out of these 11 cytotoxic regulation genes (Ly49C, Ly49I, Mapk1, Nkg2d, Shp-1, Pik3ca) in WT and mPer2  −; ; /  −; ;  entrained in light/dark (LD) or dark/dark (DD) cycles. Consistently, circadian oscillations were observed for Per1, Rev-erbα, Ly49C, and Nkg2d in WT mice under LD and DD cycles. However, these rhythmic expressions were either disrupted or dampened in mPer2  −; ; /  −; ;  mice. Comparison of gene expression between WT and mPer2  −; ; /  −; ;  mice showed that mPer2 knockout had systematically downregulated the mRNA expression of two cytotoxicity regulators, Ly49C and Nkg2d. FACS analysis further confirmed that the circadian expression of these genes was not due to the daily difference in cell numbers of NK, NKT, or T cells in bone marrow. Taken together, our results reveal that mPer2 is a critical clock component in modulating circadian rhythms in bone marrow. Furthermore, it implies that Ly49C and Nkg2d are two clock-controlled genes that may play an important role in mediating mPer2-controlled cytotoxic response. (Author correspondence: zsunusa@yahoo.com)