Isolation and enrichment of putative spermatogonial stem cells from ram (Ovis aries) testis

Binsila, Krishnan B.; Selvaraju, Sellappan; Ghosh, Subrata; Parthipan, Sivashanmugam; Archana, Santhanahalli Siddalingappa; Arunachalam, A.; Prasad, J.K.; Bhatta, Raghavendra; Ravindra, J.P.

doi:10.1016/j.anireprosci.2018.04.070

Cited by 15 publications

(17 citation statements)

References 49 publications

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“…Somatic cells (Sertoli cells, Leydig cells, myoid and peritubular cells) have an affinity towards gelatin and lectin, thus leaving SSCs in the supernatant when differentially plated. The α- and β-integrin receptors of SSCs efficiently bind to the laminin, resulting in an enrichment of SSCs through positive selection [50]. Using these approaches, SSCs have been enriched to as high as 75% in the population of donor testis cells from bull, ram or boar, summarized by Honaramooz and Yang [51].…”

Section: Approaches For Ssc Isolation Purification and Expansion Of mentioning

confidence: 99%

Manipulation of spermatogonial stem cells in livestock species

Savvulidi

Ptáček

Vargová

et al. 2019

J Animal Sci Biotechnol

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We are entering an exciting epoch in livestock biotechnology during which the fundamental approaches (such as transgenesis, spermatozoa cryopreservation and artificial insemination) will be enhanced based on the modern understanding of the biology of spermatogonial stem cells (SSCs) combined with the outstanding recent advances in genomic editing technologies and in vitro cell culture systems. The general aim of this review is to outline comprehensively the promising applications of SSC manipulation that could in the nearest future find practical application in livestock breeding. Here, we will focus on 1) the basics of mammalian SSC biology; 2) the approaches for SSC isolation and purification; 3) the available in vitro systems for the stable expansion of isolated SSCs; 4) a discussion of how the manipulation of SSCs can accelerate livestock transgenesis; 5) a thorough overview of the techniques of SSC transplantation in livestock species (including the preparation of recipients for SSC transplantation, the ultrasonographic-guided SSC transplantation technique in large farm animals, and the perspectives to improve further the SSC transplantation efficiency), and finally, 6) why SSC transplantation is valuable to extend the techniques of spermatozoa cryopreservation and/or artificial insemination. For situations where no reliable data have yet been obtained for a particular livestock species, we will rely on the data obtained from studies conducted in rodents because the knowledge gained from rodent research is translatable to livestock species to a great extent. On the other hand, we will draw special attention to situations where such translation is not possible. Electronic supplementary material The online version of this article (10.1186/s40104-019-0355-4) contains supplementary material, which is available to authorized users.

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Section: Approaches For Ssc Isolation Purification and Expansion Of mentioning

confidence: 99%

Manipulation of spermatogonial stem cells in livestock species

Savvulidi

Ptáček

Vargová

et al. 2019

J Animal Sci Biotechnol

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“…The cell culture dishes or plates used in these experiments were coated with five types of extracellular matrices. Briefly, the plates were incubated with any one of the following: 2 mg/ml gelatin (Binsila et al, 2018), 2 mg/ml laminin (Binsila et al, 2018), 2 mg/ml fibronectin (Park et al, 2018), 2 mg/ml Matrigel (Hua et al, 2011) or 2 mg/ml poly-L-lysine at 37°C, 5% CO 2 and at saturated humidity for 1 hr. Then, the coating solution was removed, and the plates were F I G U R E 2 Alkaline phosphatase staining of the cells cultured on different extracellular matrices washed with specific volumes of PBS, depending on their surface areas.…”

Section: Preparation Of the Cell Culture Platesmentioning

confidence: 99%

p53 inhibits the proliferation of male germline stem cells from dairy goat cultured on poly‐L‐lysine

Zhu

Zheng

Wang

et al. 2020

Reprod Domestic Animals

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Male germline stem cells (mGSCs) can transmit genetic materials to the next generation and dedifferentiate into pluripotent stem cells. However, in livestock, mGSC lines are difficult to establish, because of the factors that affect their isolation and culture. The extracellular matrix serves as a substrate for attachment and affects the fate of these stem cells. Poly‐L‐lysine (PL), an extracellular matrix of choice, inhibits and/or kills cancer cells, and promotes the attachment of stem cells in culture. However, how it affects the characteristics and potentials of these stem cells in culture needs to be elucidated. Here, we isolated, enriched and cultured dairy goat mGSCs on five types of extracellular matrices. To explore the best extracellular matrix to use for culturing them, the characteristics and proliferation ability of the cells were determined. Results showed that the cells shared several characteristics with previously reported mGSCs, including the poor effect of PL on their proliferative and colony‐forming abilities. Further examination showed upregulation of p53 expression in these cells, which could be inhibiting their proliferation. When a p53 inhibitor was included in the culture medium, it was confirmed to be responsible for the inhibition of proliferation in mGSCs. Optimal concentration of the inhibitor in the culture of these cells was 5 µM. Furthermore, addition of the p53 inhibitor increased the expression of the markers of self‐renewal and cell cycle in goat mGSCs. In summary, suppressing p53 is beneficial for the proliferation of dairy goat mGSCs, cultured on PL.

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“…A milestone has been reached in primates, with the successful production of offspring from cryopreserved testes grafted onto autologous rhesus monkeys [5]. However, in livestock the application of SSC technology is advancing with different pace [1,6]. The possible reasons for the slow progress of SSCs technology in livestock are inadequate methodologies in obtaining purified SSCs from the testicular cells, lack of identified SSCs markers in livestock, and non-availability of defined culture media for sustaining stemness during long-term proliferation in vitro [7,8].…”

Section: Introductionmentioning

confidence: 99%

“…Isolation of SSCs free from differentiating germ cells and somatic cells in the testicular isolate is a prerequisite for developing a suitable culture system. The population of SSCs in testicular cells isolate is extremely low, for example, 0.02-0.03% in mice [9,10] and 0.18-0.47% in adult sheep [6]. Single enrichment protocols with laminin in laboratory animals [11] and laminin in combination with bovine serum albumin (BSA) in sheep [6] were used for enriching SSCs.…”

Section: Introductionmentioning

confidence: 99%

“…The population of SSCs in testicular cells isolate is extremely low, for example, 0.02-0.03% in mice [9,10] and 0.18-0.47% in adult sheep [6]. Single enrichment protocols with laminin in laboratory animals [11] and laminin in combination with bovine serum albumin (BSA) in sheep [6] were used for enriching SSCs. The purity of SSCs has been improved when the enrichment methods were combined and adopted.…”

Section: Introductionmentioning

confidence: 99%

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EGF, GDNF, and IGF-1 influence the proliferation and stemness of ovine spermatogonial stem cells in vitro

Binsila

Selvaraju

Ghosh

et al. 2020

J Assist Reprod Genet

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Purpose The objective of the present study was to purify sheep spermatogonial stem cells (SSCs) from testicular isolate using combined enrichment methods and to study the effect of growth factors on SSC stemness during culture. Methods The testicular cells from prepubertal male sheep were isolated, and SSCs were purified using Ficoll gradients (10 and 12%) followed by differential plating (laminin with BSA). SSCs were cultured with StemPro®-34 SFM, additives, and FBS for 7 days. The various doses (ng/ml) of growth factors, EGF at 10, 15, and 20, GDNF at 40, 70, and 100 and IGF-1 at 50, 100, and 150 were tested for the proliferation and stemness of SSCs in vitro. The stemness in cultured cells was assessed using SSC markers PLZF, ITGA6, and GFRα1. Results Ficoll density gradient separation significantly (p < 0.05) increased the percentage of SSCs in 12% fraction (35.1 ± 3.8 vs 11.2 ± 3.7). Subsequently, purification using laminin with BSA plating further enriched SSCs to 61.7 ± 4.7%. GDNF at 40 ng/ml, EGF at 15 and 20 ng/ml and IGF1 at 100 and 150 ng/ml significantly (p < 0.05) improved proliferation and stemness of SSCs up to 7 days in culture. GDNF at 40 ng/ml outperformed other growth factors tested and could maintain the ovine SSCs proliferation and stemness for 36 days. Conclusions The combined enrichment method employing density gradient centrifugation and laminin with BSA plating improves the purification efficiency of ovine SSCs. GDNF at 40 ng/ml is essential for optimal proliferation and sustenance of stemness of ovine SSCs in vitro.

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Isolation and enrichment of putative spermatogonial stem cells from ram (Ovis aries) testis

Cited by 15 publications

References 49 publications

Manipulation of spermatogonial stem cells in livestock species

Manipulation of spermatogonial stem cells in livestock species

p53 inhibits the proliferation of male germline stem cells from dairy goat cultured on poly‐L‐lysine

EGF, GDNF, and IGF-1 influence the proliferation and stemness of ovine spermatogonial stem cells in vitro

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