Generation of Porcine Induced Neural Stem Cells Using the Sendai Virus

The reprogramming of cells into induced neural stem cells (iNSCs), which are faster and safer to generate than induced pluripotent stem cells, holds tremendous promise for fundamental and frontier research, as well as personalized cell-based therapies for neurological diseases. However, reprogramming cells with viral vectors increases the risk of tumor development due to vector and transgene integration in the host cell genome. To circumvent this issue, the Sendai virus (SeV) provides an alternative integration-free reprogramming method that removes the danger of genetic alterations and enhances the prospects of iNSCs from bench to bedside. Since pigs are among the most successful large animal models in biomedical research, porcine iNSCs (piNSCs) may serve as a disease model for both veterinary and human medicine. Here, we report the successful generation of piNSC lines from pig fibroblasts by employing the SeV. These piNSCs can be expanded for up to 40 passages in a monolayer culture and produce neurospheres in a suspension culture. These piNSCs express high levels of NSC markers (PAX6, SOX2, NESTIN, and VIMENTIN) and proliferation markers (KI67) using quantitative immunostaining and western blot analysis. Furthermore, piNSCs are multipotent, as they are capable of producing neurons and glia, as demonstrated by their expressions of TUJ1, MAP2, TH, MBP, and GFAP proteins. During the reprogramming of piNSCs with the SeV, no induced pluripotent stem cells developed, and the established piNSCs did not express OCT4, NANOG, and SSEA1. Hence, the use of the SeV can reprogram porcine somatic cells without first going through an intermediate pluripotent state. Our research produced piNSCs using SeV methods in novel, easily accessible large animal cell culture models for evaluating the efficacy of iNSC-based clinical translation in human medicine. Additionally, our piNSCs are potentially applicable in disease modeling in pigs and regenerative therapies in veterinary medicine.

Download Full-text

Generation of Integration-Free Porcine Induced Neural Stem Cells Using Sendai virus

10.21203/rs.3.rs-948414/v1 ◽

2021 ◽

Author(s):

Warunya Chakritbudsabong ◽

Ruttachuk Rungsiwiwut ◽

Ladawan Sariya ◽

Phakhin Juntahirun ◽

Nattarun Chaisilp ◽

...

Keyword(s):

Stem Cells ◽

Neural Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Disease Modeling ◽

Sendai Virus ◽

Large Animal ◽

Large Animal Models ◽

Induced Pluripotent ◽

Induced Neural Stem Cells

Abstract Background The reprogramming of cells to induced neural stem cells (iNSCs), faster and safer to generate than induced pluripotent stem cells, holds tremendous promise for disease modeling and personalized cell-based therapies for neurological diseases. Porcine iNSCs (piNSCs) may serve as a disease model for human medicine, as pigs are one of the most successful large animal models in biomedical research. Thus, this study aimed to establish safe and efficient integration-free piNSC lines.Methods The integration-free piNSC lines were generated by reprogramming porcine fibroblasts using the Sendai virus (SeV).Results Here we report the successful generation of integration-free piNSC lines using the SeV, with a reprogramming efficiency of 0.4%. The piNSCs can be expanded for up to 40 passages and express high levels of NSC markers (PAX6, NESTIN, and SOX2). They can produce neurons and glia, expressing TUJ, MAP2, TH, and GFAP. No induced pluripotent stem cells developed during reprogramming, and the established piNSCs did not express OCT4. Hence, the SeV can reprogram porcine fibroblast without first going through an intermediate pluripotent stage.Conclusions With the SeV approach, we generated integration-free piNSCs that may be used to assess the efficacy and safety of iNSC-based clinical translation in humans.

Download Full-text

A non-invasive method to generate induced pluripotent stem cells from primate urine

Scientific Reports ◽

10.1038/s41598-021-82883-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Johanna Geuder ◽

Lucas E. Wange ◽

Aleksandar Janjic ◽

Jessica Radmer ◽

Philipp Janssen ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Sendai Virus ◽

Cell Types ◽

Urine Samples ◽

Comparative Approach ◽

Non Invasive ◽

Human Ipscs ◽

Induced Pluripotent

AbstractComparing the molecular and cellular properties among primates is crucial to better understand human evolution and biology. However, it is difficult or ethically impossible to collect matched tissues from many primates, especially during development. An alternative is to model different cell types and their development using induced pluripotent stem cells (iPSCs). These can be generated from many tissue sources, but non-invasive sampling would decisively broaden the spectrum of non-human primates that can be investigated. Here, we report the generation of primate iPSCs from urine samples. We first validate and optimize the procedure using human urine samples and show that suspension- Sendai Virus transduction of reprogramming factors into urinary cells efficiently generates integration-free iPSCs, which maintain their pluripotency under feeder-free culture conditions. We demonstrate that this method is also applicable to gorilla and orangutan urinary cells isolated from a non-sterile zoo floor. We characterize the urinary cells, iPSCs and derived neural progenitor cells using karyotyping, immunohistochemistry, differentiation assays and RNA-sequencing. We show that the urine-derived human iPSCs are indistinguishable from well characterized PBMC-derived human iPSCs and that the gorilla and orangutan iPSCs are well comparable to the human iPSCs. In summary, this study introduces a novel and efficient approach to non-invasively generate iPSCs from primate urine. This will extend the zoo of species available for a comparative approach to molecular and cellular phenotypes.

Download Full-text

Modeling CNS Involvement in Pompe Disease Using Neural Stem Cells Generated from Patient-Derived Induced Pluripotent Stem Cells

Cells ◽

10.3390/cells10010008 ◽

2020 ◽

Vol 10 (1) ◽

pp. 8

Author(s):

Yu-Shan Cheng ◽

Shu Yang ◽

Junjie Hong ◽

Rong Li ◽

Jeanette Beers ◽

...

Keyword(s):

Stem Cells ◽

Neural Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Pompe Disease ◽

Drug Efficacy ◽

Cns Involvement ◽

Glycogen Accumulation ◽

Induced Pluripotent ◽

Alpha Glucosidase

Pompe disease is a lysosomal storage disorder caused by autosomal recessive mutations in the acid alpha-glucosidase (GAA) gene. Acid alpha-glucosidase deficiency leads to abnormal glycogen accumulation in patient cells. Given the increasing evidence of central nervous system (CNS) involvement in classic infantile Pompe disease, we used neural stem cells, differentiated from patient induced pluripotent stem cells, to model the neuronal phenotype of Pompe disease. These Pompe neural stem cells exhibited disease-related phenotypes including glycogen accumulation, increased lysosomal staining, and secondary lipid buildup. These morphological phenotypes in patient neural stem cells provided a tool for drug efficacy evaluation. Two potential therapeutic agents, hydroxypropyl-β-cyclodextrin and δ-tocopherol, were tested along with recombinant human acid alpha-glucosidase (rhGAA) in this cell-based Pompe model. Treatment with rhGAA reduced LysoTracker staining in Pompe neural stem cells, indicating reduced lysosome size. Additionally, treatment of diseased neural stem cells with the combination of hydroxypropyl-β-cyclodextrin and δ-tocopherol significantly reduced the disease phenotypes. These results demonstrated patient-derived Pompe neural stem cells could be used as a model to study disease pathogenesis, to evaluate drug efficacy, and to screen compounds for drug discovery in the context of correcting CNS defects.

Download Full-text