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, 27 (2), 241-252

Long-term Propagation of Tree Shrew Spermatogonial Stem Cells in Culture and Successful Generation of Transgenic Offspring

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Long-term Propagation of Tree Shrew Spermatogonial Stem Cells in Culture and Successful Generation of Transgenic Offspring

Chao-Hui Li et al. Cell Res.

Abstract

Tree shrews have a close relationship to primates and have many advantages over rodents in biomedical research. However, the lack of gene manipulation methods has hindered the wider use of this animal. Spermatogonial stem cells (SSCs) have been successfully expanded in culture to permit sophisticated gene editing in the mouse and rat. Here, we describe a culture system for the long-term expansion of tree shrew SSCs without the loss of stem cell properties. In our study, thymus cell antigen 1 was used to enrich tree shrew SSCs. RNA-sequencing analysis revealed that the Wnt/β-catenin signaling pathway was active in undifferentiated SSCs, but was downregulated upon the initiation of SSC differentiation. Exposure of tree shrew primary SSCs to recombinant Wnt3a protein during the initial passages of culture enhanced the survival of SSCs. Use of tree shrew Sertoli cells, but not mouse embryonic fibroblasts, as feeder was found to be necessary for tree shrew SSC proliferation, leading to a robust cell expansion and long-term culture. The expanded tree shrew SSCs were transfected with enhanced green fluorescent protein (EGFP)-expressing lentiviral vectors. After transplantation into sterilized adult male tree shrew's testes, the EGFP-tagged SSCs were able to restore spermatogenesis and successfully generate transgenic offspring. Moreover, these SSCs were suitable for the CRISPR/Cas9-mediated gene modification. The development of a culture system to expand tree shrew SSCs in combination with a gene editing approach paves the way for precise genome manipulation using the tree shrew.

Figures

Figure 1
Figure 1
Cell surface marker Thy1 can be used to enrich tree shrew spermatogonial stem cells (SSCs). (A) RT-PCR detected Thy1 mRNA expression in tree shrew testis, brain, lung, and spleen. No amplification was detected in the heart, small intestine, kidney, liver, and negative control (negative Ctrl) in which template cDNA was omitted. (B) Thy1+ cell population was reproducibly obtained by FACS of a testicular tissue single-cell suspension from prepubertal (3 month, ∼16%, n = 5) and adult (1 year, ∼3%, n = 5) tree shrews (upper panel). Lower panel show the morphology of sorted Thy1+ and Thy1 cells in fluorescent and bright fields (scale bar, 100 μm). (C) mRNA of several SSC markers (Itgb1, Egr3, and Zbtb16) was prominently expressed in Thy1+ cells, whereas Ddx4 was highly expressed in Thy1 cells. Negative Ctrl was included in which template cDNA was omitted. Experiments were repeated three times with independent biological samples. (D) Immunostaining with Thy1 antibody detected the presence of Thy1+ cells at the basement membrane of the seminiferous tubules. DNA was counterstained with DAPI. Immunostaining by omitting Thy1 antibody was used as negative Ctrl (scale bar, 10 μm). Experiments were repeated three times with independent biological samples.
Figure 2
Figure 2
RNA sequencing analyses reveal the potential signaling pathways activated in tree shrew undifferentiated SSCs. (A) Relative expression of some SSC marker genes and transcription factors in Thy1+ and Thy1 cells isolated by FACS. (B) Relative expression of some key components of signaling pathways including GDNF, Wnt/β-catenin, FGF2, and Jak-STAT in Thy1+ and Thy1 cells sorted by FACS. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed the signaling pathway enrichments for genes that were upregulated and downregulated in Thy1+ cells. (D) Principle component analysis (PCA) revealed that primary Thy1+ cells (P) were separated far from Thy1+ cells cultured for different passages (P0-P2), whereas P0-P2 cells were clustered together. (E) Unbiased clustering analysis of primary (P) and cultured Thy1+ cells at differential passages (P0-P2). (F) Relative expression of several marker genes and transcription factors in primary Thy1+ cells (P) and Thy1+ cells cultured for different passages (P0-P2). (G) Gene Ontology (GO) terms and KEGG analyses revealed the terms and signaling pathways enriched for genes downregulated or upregulated during culture of Thy1+ cells (P and P0-P2). (H) Quantitative RT-PCR revealed the relative mRNA expression levels of Axin2 in primary Thy1+ (P) and Thy1 cells, and in Thy1+ cells cultured for different passages (P0, P1, and P2). Experiments were repeated three times with independent biological samples.
Figure 3
Figure 3
Establishment of tree shrew spermatogonial stem cell lines. (A) Thy1+ cells were cultured in the presence of 10 ng/mL Wnt3a and Sertoli cell feeder. Images show the appearance of germ cell clumps at passage 3 (P3, upper panel, arrow head), and stem cell colonies at P40 (lower panel) (scale bar, 100 μm). (B) Propagation dynamics of three lines of tree shrew SSCs in culture. Data are represented as mean ± SEM. (C) Schematic illustration of the culture system for tree shrew SSC expansion. (D) RT-PCR and immunostaining revealed the expression of several germ cell markers and SSC markers in long-term expanded tree shrew SSCs (scale bar, 20 μm). (E) Long-term expanded SSCs contain normal chromosome number (2n = 62).
Figure 4
Figure 4
Expanded tree shrew SSCs are capable of normal spermatogenesis and suitable for CRISPR-/Cas9-mediated gene editing. (A) Tree shrew SSCs transfected with lentiviral vectors stably expressed EGFP (scale bar, 20 μm). (B) Schematic illustration of SSC transplantation. (C) Recipient testes examined by fluorescent dissection microscopy at 0 day (D0), 40 days (D40), 100 days (D100), and 250 days (D250) post transplantation of EGFP-SSCs. Note that green seminiferous tubules were clearly visible at D40, D100, and D250. Testis ligated for 100 days was set as negative control (NC). (D) Recipient testicular tissue sections were examined by immunostaining for the expression of EGFP. At D0, no EGFP+ cells were detected within the seminiferous tubules and rare EGFP+ cells were found suspended in the lumen of seminiferous tubules (arrow head). At D40 through D250, EGFP+ germ cells at differential developmental stages were clearly detected within seminiferous tubules (scale bar, 20 μm). (E) Genomic DNA was prepared from sperm collected from epididymis of recipient tree shrew at D250 post transplantation. Nest-PCR analysis showed the insertion of EGFP transgene in sperm genome (∼200 sperm per sample), which was confirmed by sequencing PCR products. PCR reaction with genomic DNA from wide-type SSCs as templates was used as NC, whereas PCR reaction with genomic DNA from EGFP-SSCs as templates was set as positive control (PC). (F) Genomic DNA was prepared from offspring generated by recipient males. PCR amplification of integrated transgene was conducted with two different primer pairs and PCR products were sequenced to confirm the integration of transgene. Genomic DNA from wild-type (WT) tree shrew was used as NC. Genomic DNA from EGFP-SSCs was used as positive control 1 (PC1) and EGFP-lentiviral expressing plasmids were used as positive control 2 (PC2). (G) Immunoblotting analysis confirmed the expression of EGFP proteins in several tissues collected from one transgenic pup. Protein from WT SSCs was used as NC. Proteins from EGFP-SSCs were used as PC. (H) Fluorescent image showing the EGFP expression in the transgenic tree shrew (left) versus the control tree shrew (right). (I) CRISPR-/Cas9-mediated targeting of the App gene in tree shrew SSCs. DNA sequencing of PCR products confirmed the targeted mutations in the App gene. Mutation type 1 has an insertion of “A” at the target site, and mutation type 2 contains a 25-bp deletion at the target site.

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