A Nonhuman Primate Model of Lung Regeneration: Detergent-Mediated Decellularization and Initial In Vitro Recellularization with Mesenchymal Stem Cells

Abstract Preclinical animal models are important for evaluating the safety and therapeutic efficacy of new therapeutic modalities such as gene therapy. From the different large animal models, nonhuman primate models have emerged over the last decades as highly desirable experimental systems from both a pathophysiologic and pharmacokinetic viewpoint and the study of nonhuman primates has provided important information on the efficacy and safety of gene therapy systems in vivo prior to human trials. The common marmoset (Callithrix jacchus) has the advantage that it is a small, and thus relatively inexpensive nonhuman primate model. Currently, very little data on the transduction efficiency of foamyviral vectors for gene transfer into marmoset stem cells exists. We therefore performed a direct comparison using identically designed gammaretroviral, lentiviral and foamyviral vector constructs expressing the enhanced green fluorescent protein (EGFP) from the spleen focus forming virus (SFFV) promoter pseudotyped with either the modified human foamy virus (HFV) envelope EM140 or the G-protein of vesicular stomatitis virus (VSV-G) for the transduction of common marmoset embryonic stem cells (CMES) as well as marmoset CD34+ hematopoietic progenitor cells. Virus stocks of these vectors were prepared by polyethyleneimine-mediated transfection of 293T cells and concentrated approximately 10-fold by centrifugation for 4 hours at 10.000 g at 4°C. Three different target cell populations were transduced: common marmoset embryonic stem cells (CMES) or cryopreserved CD34-enriched cells from bone marrow of a common marmoset either after a two-day prestimulation in the presence of IL-6, FLT3L, cSCF and TPO at a concentration of 100 ng/mL each, or after overnight incubation with 100 ng/mL SCF only. Equal numbers of cells were exposed to the four different vector preparations for 14 hours in 12-well dishes coated with CH-296. The read-out was based on fluorescence microscopy of colonies plated in methyl cellulose as well as flow cytometry (FACS). Foamyviral vectors with the foamyviral envelope were the most efficient gene transfer tool for marmoset hematopoietic CD34-positive cells with stable transduction rates of over 80% as assessed by flow cytometry at both 2 or 7 days after the end of transduction and on average 88% transduction efficiency into colony forming cells (CFU-C). Transduction of CFU-C with all other vector preparations was below 60%. In CMES, initial gene transfer rates of over 80% were achieved with the VSV-G pseudotype lentiviral vector, however, expression decreased to 13% after 7 days. In contrast, the foamyviral vector pseudotyped with the foamyviral envelope decreased only from 49% to 24% after 7 days. In conclusion, we achieved stable viral gene transfer and expression in CMES cells as well as highly efficient gene transfer into common marmoset hematopoietic CD34 positive cells using foamyviral vectors. These results suggest that foamyviral vectors may be highly feasible vectors for stem cell gene transfer and thus set the stage for a more detailed analysis of this vector system in transplantation studies in this nonhuman primate model.

Download Full-text

Proteomic Insights into Senescence of Testicular Peritubular Cells from a Nonhuman Primate Model

Cells ◽

10.3390/cells9112498 ◽

2020 ◽

Vol 9 (11) ◽

pp. 2498

Author(s):

Jan B. Stöckl ◽

Nina Schmid ◽

Florian Flenkenthaler ◽

Charis Drummer ◽

Rüdiger Behr ◽

...

Keyword(s):

Nonhuman Primate ◽

Human Testis ◽

Nonhuman Primate Model ◽

Primate Model ◽

Morphological Alterations ◽

Age Related ◽

Cell Niche ◽

Peritubular Cells

Age-related changes in the human testis may include morphological alterations, disturbed steroidogenesis, and impaired spermatogenesis. However, the specific impact of cell age remains poorly understood and difficult to assess. Testicular peritubular cells fulfill essential functions, including sperm transport, contributions to the spermatogonial stem cell niche, and paracrine interactions within the testis. To study their role in age-associated decline of testicular functions, we performed comprehensive proteome and secretome analyses of repeatedly passaged peritubular cells from Callithrix jacchus. This nonhuman primate model better reflects the human testicular biology than rodents and further gives access to young donors unavailable from humans. Among 5095 identified proteins, 583 were differentially abundant between samples with low and high passage numbers. The alterations indicate a reduced ability of senescent peritubular cells to contract and secrete proteins, as well as disturbances in nuclear factor (NF)-κB signaling and a reduced capacity to handle reactive oxygen species. Since this in vitro model may not exactly mirror all molecular aspects of in vivo aging, we investigated the proteomes and secretomes of testicular peritubular cells from young and old donors. Even though the age-related alterations at the protein level were less pronounced, we found evidence for impaired protein secretion, altered NF-κB signaling, and reduced contractility of these in vivo aged peritubular cells.

Download Full-text