Enhanced Enrichment of Medaka Ovarian Germline Stem Cells by a Combination of Density Gradient Centrifugation and Differential Plating

Abstract

Fish ovarian germline stem cells (OGSCs) have great potential in various biological fields due to their ability to generate large numbers of mature eggs. Therefore, selective enrichment of OGSCs is a prerequisite for successful applications. To determine the optimal conditions for the enrichment of OGSCs from Japanese medaka (Oryzias latipes), we evaluated the effects of Percoll density gradient centrifugation (PDGC), differential plating (DP), and a combination of both methods. Based on cell morphology and gene expression of germ cell-specific Vasa and OGSC-specific Nanos2, we demonstrated that of seven density fractions obtained following PDGC, the 30–35% density fraction contained the highest proportion of OGSCs, and that Matrigel was the most effective biomolecule for the enrichment of Oryzias latipes OGSCs by DP in comparison to laminin, fibronectin, gelatin, and poly-l-lysine. Furthermore, we confirmed that PDGC and DP in combination significantly enhanced the efficiency of OGSC enrichment. The enriched cells were able to localize in the gonadal region at a higher efficiency compared to non-enriched ovarian cells when transplanted into the developing larvae. Our approach provides an efficient way to enrich OGSCs without using OGSC-specific surface markers or transgenic strains expressing OGSC-specific reporter proteins.

Advantages, Factors, Obstacles, Potential Solutions, and Recent Advances of Fish Germ Cell Transplantation for Aquaculture—A Practical Review

“…After centrifugation, desired cells harboring a specific range of densities, which form bands between Percoll layers, are harvested for transplantation. For example, in zebrafish and medaka, as GSCs were abundant in 25–35% density Percoll fractions, cells harvested from these fractions were subjected to transplantation [ 69 , 70 ].…”

“…Enrichment methods can be used solely or combinedly. By combining them, the limitation of each method can be mitigated, which would lead to higher purities than those of enrichment with a single method [ 70 ]. The improved purity of GSCs after enrichment resulted in an increase of colonization efficiencies (up to a 17.5-fold increase) compared to those of unsorted cells [ 19 , 20 , 62 , 70 , 73 , 86 ].…”

“…By combining them, the limitation of each method can be mitigated, which would lead to higher purities than those of enrichment with a single method [ 70 ]. The improved purity of GSCs after enrichment resulted in an increase of colonization efficiencies (up to a 17.5-fold increase) compared to those of unsorted cells [ 19 , 20 , 62 , 70 , 73 , 86 ]. Improved colonization rates by enriching donor cells will contribute to efficient surrogate offspring production, reducing efforts for screening works.…”

Who is the best surrogate for germ stem cell transplantation in fish?

“…Since surrogate reproduction in fish is a long term and laborious effort, whole technology needs to be optimized to maximize its success. Optimal conditions for GSCs isolation (Shikina et al, 2013), purification (Ryu and Gong, 2020), and in vitro expansion (Iwasaki-Takahashi et al, 2020; Xie et al, 2019) were identified. Several studies already paid attention to the investigation of variables related to used recipients.…”

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“…Em contraste a estes resultados, Ryu & Gong, (2020) observaram que os genes nanos2 e vasa apresentaram altos níveis de expressão gênica nas oogôniastronco submetidas ao plaqueamento diferencial. Este fato sugere que as condições de purificação, tal como meio, ação enzimática e período de purificação podem ter afetado a qualidade da célula.…”