Initial germ cell to somatic cell ratio impacts the efficiency of SSC expansion <i>in vitro</i>

Gat, Itai; Maghen, Leila; Filice, Melissa; Kenigsberg, Shlomit; Wyse, Brandon A.; Zohni, Khaled; Saraz, Peter; Fisher, Andrée Gauthier; Librach, Clifford L.

doi:10.1080/19396368.2017.1406013

Cited by 4 publications

(3 citation statements)

References 45 publications

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“…Enrichment of cell suspensions in SSCs is also possible using several methods, such as cell sorting followed by in vitro expansion [ 111 ] or differential plating [ 112 ]. However, these techniques still need to be refined because of partial results in terms of cell purity [ 113 ] and viability.…”

Section: Resultsmentioning

confidence: 99%

Tissue Engineering to Improve Immature Testicular Tissue and Cell Transplantation Outcomes: One Step Closer to Fertility Restoration for Prepubertal Boys Exposed to Gonadotoxic Treatments

Vento

Vermeulen

Michele

et al. 2018

IJMS

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Despite their important contribution to the cure of both oncological and benign diseases, gonadotoxic therapies present the risk of a severe impairment of fertility. Sperm cryopreservation is not an option to preserve prepubertal boys’ reproductive potential, as their seminiferous tubules only contain spermatogonial stem cells (as diploid precursors of spermatozoa). Cryobanking of human immature testicular tissue (ITT) prior to gonadotoxic therapies is an accepted practice. Evaluation of cryopreserved ITT using xenotransplantation in nude mice showed the survival of a limited proportion of spermatogonia and their ability to proliferate and initiate differentiation. However, complete spermatogenesis could not be achieved in the mouse model. Loss of germ cells after ITT grafting points to the need to optimize the transplantation technique. Tissue engineering, a new branch of science that aims at improving cellular environment using scaffolds and molecules administration, might be an approach for further progress. In this review, after summarizing the lessons learned from human prepubertal testicular germ cells or tissue xenotransplantation experiments, we will focus on the benefits that might be gathered using bioengineering techniques to enhance transplantation outcomes by optimizing early tissue graft revascularization, protecting cells from toxic insults linked to ischemic injury and exploring strategies to promote cellular differentiation.

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Section: Resultsmentioning

confidence: 99%

Tissue Engineering to Improve Immature Testicular Tissue and Cell Transplantation Outcomes: One Step Closer to Fertility Restoration for Prepubertal Boys Exposed to Gonadotoxic Treatments

Vento

Vermeulen

Michele

et al. 2018

IJMS

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“…The percentages of PLZF, ITGA6, and GFRα1 positive cells in the SSCs cultured for 7 days with different doses of growth factors namely, epidermal growth factor at 10 (E10), 15 (E15), and 20 (E20) ng/ml; glial cell line-derived neurotrophic growth factor at 40 (G40), 70 (G70), and 100 (G100) ng/ml and insulinlike growth factor-1 at 50 (I50), 100 (I100), and 150 (I150) ng/ml. n Values did not differ significantly between the control and the treatment groups were carried out successfully with various levels of purified SSCs, 26% [39] and 91% [11] in rodents, 70-73% [40,41] in porcine, 72% in bovine [42], and 83% in goat [43]. The variation in the purity of the SSC obtained in the present study as compared to previous studies may be attributed to the developmental stage of the testes used for isolation, differences in enrichment protocol (single or double) employed and markers used for validation [13].…”

Section: Discussionmentioning

confidence: 99%

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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“…35,47 Indeed, the importance of the germ-versus somatic-cell ratio in culture was demonstrated, showing an impact on SSC proliferation. 52,53 The influence of the medium was also pinpointed when Gat et al observed more germ-cell aggregate formation when using DMEM/ F12 instead of StemPro-34. 54 Others also examined the efficiency of differential plating to select germ cells from TCSs, but did not find a difference in germ-cell numbers recovered from whole TCSs and differentially plated cells after 14 days of culture.…”

Section: Ssc Propagationmentioning

confidence: 99%

<p>Role of stem cells in fertility preservation: current insights</p>

Vermeulen¹,

Giudice²,

Vento³

et al. 2019

SCCAA

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While improvements made in the field of cancer therapy allow high survival rates, gonadotoxicity of chemo- and radiotherapy can lead to infertility in male and female pre- and postpubertal patients. Clinical options to preserve fertility before starting gonadotoxic therapies by cryopreserving sperm or oocytes for future use with assisted reproductive technology (ART) are now applied worldwide. Cryopreservation of pre- and postpubertal ovarian tissue containing primordial follicles, though still considered experimental, has already led to the birth of healthy babies after autotransplantation and is performed in an increasing number of centers. For prepubertal boys who do not produce gametes ready for fertilization, cryopreservation of immature testicular tissue (ITT) containing spermatogonial stem cells may be proposed as an experimental strategy with the aim of restoring fertility. Based on achievements in nonhuman primates, autotransplantation of ITT or testicular cell suspensions appears promising to restore fertility of young cancer survivors. So far, whether in two- or three-dimensional culture systems, in vitro maturation of immature male and female gonadal cells or tissue has not demonstrated a capacity to produce safe gametes for ART. Recently, primordial germ cells have been generated from embryonic and induced pluripotent stem cells, but further investigations regarding efficiency and safety are needed. Transplantation of mesenchymal stem cells to improve the vascularization of gonadal tissue grafts, increase the colonization of transplanted cells, and restore the damaged somatic compartment could overcome the current limitations encountered with transplantation.

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Initial germ cell to somatic cell ratio impacts the efficiency of SSC expansion in vitro

Abstract: SSC: spermatogonial stem cells; DP: differential plating; NOA: non-obstructive azoospermia; MACS: magnetic-activated cells sorting; FACS: fluorescence-activated cells sorting.

Cited by 4 publications

References 45 publications

Tissue Engineering to Improve Immature Testicular Tissue and Cell Transplantation Outcomes: One Step Closer to Fertility Restoration for Prepubertal Boys Exposed to Gonadotoxic Treatments

Tissue Engineering to Improve Immature Testicular Tissue and Cell Transplantation Outcomes: One Step Closer to Fertility Restoration for Prepubertal Boys Exposed to Gonadotoxic Treatments

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

<p>Role of stem cells in fertility preservation: current insights</p>

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