Characteristics of spermatogonial stem cells derived from neonatal porcine testis

Shi, Ruyi; Bai, Yinshan; Li, S.; Wei, Hengxi; Zhang, X.; Li, L.; Tian, Xiue; Jiang, Qi; Wang, Chong; Qin, Lili; Cai, Jun; Zhang, S.

doi:10.1111/and.12327

Cited by 9 publications

(13 citation statements)

References 44 publications

(62 reference statements)

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“…SSCs specifically express several genes, notably integrin α6 , integrinβ1 , CD9 , CD24 , SOX2 , and Oct4 . In contrast, they also fail to express other genes: Sca , c-kit , CD34 , MHC-I , and CD51 [ 37 , 38 , 39 , 40 , 41 , 42 ]. For pigs, PGP9.5 , ZBTB16 , CD90 , GFRα1 , NANOG2 , POU5F1 , SSEA1 have been proved as molecular markers of spermatogonia in testes [ 9 , 43 , 44 , 45 , 46 , 47 , 48 ].…”

Section: Discussionmentioning

confidence: 99%

Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes

Wang

Chen

Zhang

et al. 2015

IJMS

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Spermatogonial stem cells (SSCs) renew themselves throughout the life of an organism and also differentiate into sperm in the adult. They are multipopent and therefore, can be induced to differentiate into many cells types in vitro. SSCs from pigs, considered an ideal animal model, are used in studies of male infertility, regenerative medicine, and preparation of transgenic animals. Here, we report on a culture system for porcine SSCs and the differentiation of these cells into neuron-like cells and adipocytes. SSCs and Sertoli cells were isolated from neonatal piglet testis by differential adhesion and SSCs were cultured on a feeder layer of Sertoli cells. Third-generation SSCs were induced to differentiate into neuron-like cells by addition of retinoic acid, β-mercaptoethanol, and 3-isobutyl-1-methylxanthine (IBMX) to the induction media and into adipocytes by the addition of hexadecadrol, insulin, and IBMX to the induction media. The differentiated cells were characterized by biochemical staining, qRT-PCR, and immunocytochemistry. The cells were positive for SSC markers, including alkaline phosphatase and SSC-specific genes, consistent with the cells being undifferentiated. The isolated SSCs survived on the Sertoli cells for 15 generations. Karyotyping confirmed that the chromosomal number of the SSCs were normal for pig (2n = 38, n = 19). Pig SSCs were successfully induced into neuron-like cells eight days after induction and into adipocytes 22 days after induction as determined by biochemical and immunocytochemical staining. qPCR results also support this conclusion. The nervous tissue markers genes, Nestin and β-tubulin, were expressed in the neuron-like cells and the adipocyte marker genes, PPARγ and C/EBPα, were expressed in the adipocytes.

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Section: Discussionmentioning

confidence: 99%

Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes

Wang

Chen

Zhang

et al. 2015

IJMS

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“…Reverse transcription-PCR was performed using a cDNA synthesis kit (Promega, Madison, WI, USA) and 2 μg of total RNA according to the manufacturer's protocol. The SSC related genes (GFRα1, PGP9.5, Plzf and NANOS2 [28]), SSC differentiation-related genes (Stra8, c-kit, RARγ, and cytochrome P450 family 26 enzymes B1 (CYP26B1)), an anti-apoptosis gene (Bcl2), genes with post-meiotic expression (transition protein 1 (Tnp1) and protamine 1 (Prm1)), and histone acetylation-related genes (Cdyl and Hdac1) were detected by RT-PCR. β-actin was used as an internal control.…”

Section: Methodsmentioning

confidence: 99%

Retinoic acid promotes in vitro development of haploid germ cells from pre-pubertal porcine spermatogenic cells

Kang

Zhang

et al. 2019

Preprint

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Spermatogonial stem cells (SSCs) self-renew and contribute genetic information to the next generation. Inducing directional differentiation of porcine SSCs may be an important strategy in exploring the mechanisms of spermatogenesis and developing better treatment methods for male sterility. Here, we established an in vitro culture model for porcine small seminiferous tubule segments, to induce SSCs to differentiate into single-tail haploid spermatozoa. The culture model subsequently enabled spermatozoa to express the sperm-specific protein acrosin, and oocytes to develop to blastocyst stage after round spermatid injection. The addition of retinoic acid (RA) to the differentiation media promoted the efficiency of haploid differentiation. RT-PCR analysis indicated that RA stimulated the expression of Stra8 but reduced the expression of NANOS2 in spermatogonia. Genes involved in post-meiotic development, Prm1 and Tnp1, were up-regulated in the presence of RA. The addition of RAR inhibitor, BMS439, showed that RA enhanced the expression of cAMP responsive-element binding protein through RAR, and promoted the formation of round spermatids.

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“…Testes from 5-day-old piglets (Landrace) were provided by Guangdong Wens Foodstuff Co., Ltd. (Yunfu, China). The porcine testicular cells were prepared and cultured as previously described [ 17 ]. The unattached testicular cells were gently transferred into a new 10-cm culture dish by differential plating culture, repeated hourly for five transfers.…”

Section: Methodsmentioning

confidence: 99%

“…The unattached testicular cells were gently transferred into a new 10-cm culture dish by differential plating culture, repeated hourly for five transfers. When the majority of the remaining suspended cells were putative porcine mGSCs, density gradient centrifugation on Percoll (Pharmacia) was performed as previously described [ 17 ]. After centrifugation, lymphocytes and cellular debris were in the top layer, erythrocytes were in the bottom layer, and the target cells were in the middle layer.…”

Section: Methodsmentioning

confidence: 99%

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Previously claimed male germline stem cells from porcine testis are actually progenitor Leydig cells

et al. 2018

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BackgroundMale germline stem cells (mGSCs) offer great promise in regenerative medicine and animal breeding due to their capacity to maintain self-renewal and to transmit genetic information to the next generation following spermatogenesis. Human testis-derived embryonic stem cell-like cells have been shown to possess potential of mesenchymal progenitors, but there remains confusion about the characteristics and origin of porcine testis-derived stem cells.MethodsPorcine testis-derived stem cells were obtained from primary testicular cultures of 5-day old piglets, and selectively expanded using culture conditions for long-term culture and induction differentiation. The stem cell properties of porcine testis-derived stem cells were subsequently assessed by determining the expression of pluripotency-associated markers, alkaline phosphatase (AP) activity, and capacity for sperm and multilineage differentiation in vitro. The gene expression profile was compared via microarray analysis.ResultsWe identified two different types of testis-derived stem cells (termed as C1 and C2 here) during porcine testicular cell culture. The gene expression microarray analysis showed that the transcriptome profile of C1 and C2 differed significantly from each other. The C1 appeared to be morphologically similar to the previously described mouse mGSCs, expressed pluripotency- and germ cell-associated markers, maintained the paternal imprinted pattern of H19, displayed alkaline phosphatase activity, and could differentiate into sperm. Together, these data suggest that C1 represent the porcine mGSC population. Conversely, the C2 appeared similar to the previously described porcine mGSCs with three-dimensional morphology, abundantly expressed Leydig cell lineage and mesenchymal cell-specific markers, and could differentiate into testosterone-producing Leydig cells, suggesting that they are progenitor Leydig cells (PLCs).ConclusionCollectively, we have established the expected characteristics and markers of authentic porcine mGSCs (C1). We found for the first time that, the C2, equivalent to previously claimed porcine mGSCs, are actually progenitor Leydig cells (PLCs). These findings provide new insights into the discrepancies among previous reports and future identification and analyses of testis-derived stem cells.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-0931-0) contains supplementary material, which is available to authorized users.

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Characteristics of spermatogonial stem cells derived from neonatal porcine testis

Cited by 9 publications

References 44 publications

Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes

Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes

Retinoic acid promotes in vitro development of haploid germ cells from pre-pubertal porcine spermatogenic cells

Previously claimed male germline stem cells from porcine testis are actually progenitor Leydig cells

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