Mesenchymal stem cells in the dental pulp exhibit a tendency for differentiation into various dental lineages and hold great potential as a major conduit for regenerative treatment in dentistry. Although they can be readily isolated from teeth, the exact characteristics of these stem cells have not been fully understood so far. When compared to twodimensional (2D) cultures, three-dimensional (3D) cultures have the advantage of enriching the stem cell population. Hence, 3D-organoid culture and 3D-sphere culture were applied to dental pulp cells in the current study. Although the establishment of the organoid culture proved unsuccessful, the 3D-sphere culture readily initiated the stable generation of cell aggregates, which continued to grow and could be passaged to the second round. Interestingly, a significant increase in SOX2 expression was detected in the 3D-spheroid culture compared to the 2D culture. These results indicate the enrichment of the stemness-high population in the 3D-sphere culture. Thus, 3D-sphere culture may act as a link between the conventional and 3D-organoid cultures and aid in understanding the characteristics of dental pulp stem cells.

본 연구는 동해유입하천 (강릉 연곡천, 양양 남대천), 한강수계 (섬강, 속사천), 낙동강수계 (길안천)에 서식하는 참갈겨니 (Zacco koreanus) 개체군을 대상으로 채집된 110개 체로부터 미토콘드리아 DNA COI 유전자 (mitochondrial DNA cytochrome oxidase I)를 분자마커로 이용하여 계통지리학적 분석을 수행하고, 추가적으로 강릉 연곡천 상·중· 하류 개체군을 대상으로 집단유전학적 분석을 수행하였다. 계통지리학 분석 결과, 동해유입하천과 한강수계의 참갈겨니 개체군은 동일한 단일계통을 나타내었고, 낙동강수계의 개체군은 상이한 계통으로 분기됨을 나타내었으며, 다른 수계 계통과의 유전적 거리 수치 범위가 평균 4.0% (3.7~4.2%)로서 동일종 이상 수준을 보여 잠재종 가능성을 시사하였다. 참갈겨니가 서식하는 수계에 따른 형태학적 차이는 연구된 바 있으나 DNA 염기서열의 변이를 이용한 분자유전학적 연구는 부족한 실정이므로 본 연구 결과는 향후 낙동강수계 참갈겨니 개체군의 계통분류학적 연구에 기초자료로 활용될 수 있을 것으로 판단된다. 추후 집단유전체학 및 생태학적 분석을 통하여 관찰된 낙동강수계 계통이 다른 종, 잠재종 혹은 단순히 큰 수준의 종내 변이를 나타내는지에 대한 추가적인 연구가 필요하다. 강릉 연곡천 상·중·하류에 서식하는 개체군의 집단유전학 분석을 통해 중류의 개체군이 상대적으로 높은 다양성을 나타냈으며 상·중·하류 개체군 간의 유전적 차이는 나타나지 않았다. 이는 상·중·하류 개체군 간 유전자 확산이 원활하게 이루어지고 있음을 의미하며 하천의 개체군 간 연결성을 판단할 수 있는 지표로 활용될 수 있다. 하지만 생태학적 시간 스케일의 연구에 더 적합한 분자마커를 이용한 추후 연구가 필요할 것으로 사료된다.

The mesenchymal stem cells (MSCs) that reside in dental tissues hold a great potential for future applications in regenerative dentistry. In this study, we used human dental pulp cells, isolated from the molars (DPCs), in order to establish the organoid culture. DPCs were established after growing pulp cells in an MSC expansion media (MSC-EM). DPCs were subjected to organoid growth media (OGM) in comparison with human dental pulp stem cells (DPSCs). Inside the extracellular matrix in the OGM, the DPCs and DPSCs readily formed vessel-like structures, which were not observed in the MSC-EM. Immunocytochemistry analysis and flow cytometry analysis showed the elevated expression of CD31 in the DPCs and DPSCs cultured in the OGM. These results suggest endothelial cell-prone differentiation of the DPCs and DPSCs in organoid culture condition.

Brachymystax lenok tsinlingensis (family Salmonidae), cold freshwater fish, is endemic to Asia. This species is currently distributed throughout Russia, Mongolia, China and the Korean Peninsula. B. lenok tsinlingensis in South Korea was severely affected by anthropogenic activities such as habitat destruction, agricultural run-off and water pollution, and hence this fish has recently been dramatically decreased in its population sizes and become now critically endangered. To recover the number of individuals of B. lenok tsinlingensis, stocking or translocation programs have been conducted continuously by local governments since 1970s. However, these programs made little effort to clarify populations that may have originated from stocked, translocated or introduced fish. An understanding of genetic characteristics of endangered populations is critical to develop effective conservation and restoration plans especially because genetic diversity ensues their future fate. Therefore, we assessed the “conservation status” of this species by estimating the level of genetic diversity and genetic structure among ten geographic populations including restored populations via reinforcement and supplementation. Also, we aimed to trace the genetic origins of the newly translocated population (Chiak) through a restoration practice program. Moreover, we inferred the phylogenetic relationships among Korean lenok populations as well as across the Northeast Asia. Two hundred eighteen individuals of B. lenok tsinlingensis were sampled from ten localities (Yanggu, Injae, Seorak, Bangtae and Hongcheon: North Han River basin; Pyeongchang, Chiak and Jeongseon: South Han River basin; Taebaek and Bonghwa: Nakdong River basin in South Korea). Based on mitochondrial DNA (mtDNA) control region and eight nuclear microsatellite loci, we found extremely low levels of within-population genetic diversity, which suggests small effective population sizes (Ne) within populations. For mtDNA control region, each population housed one, or at most, two haplotypes that are restricted to the respective localities, meaning that these ‘genetically unique’ lineages will be lost permanently if the local populations undergo extinction. The overall values of haplotype diversity (h) and nucleotide diversity (π) for the entire Korean population were 0.703 ± 0.024 and 0.021 ± 0.010, respectively. In the case of microsatellites, average number of alleles across the eight loci for the entire population was 9.1 and allelic richness (AR) per population ranged from 2.375 to 4.144 (mean = 3.104). The values of observed heterozygosity (HO) and expected heterozygosity (HE) were similar to each other [HO: 0.400 ~ 0.590 (mean = 0.518); HE: 0.407 ~ 0.608 (mean = 0.504)]. The inbreeding coefficient (FIS) values were generally low, ranging from 0.048 to 0.279. Consequently, the majority of the populations (except Yanggu and Pyeongchang) were not significantly deviated from Hardy-Weinberg equilibrium (HWE), suggesting random mating at these loci tested. In addition, we found that Korean lenok populations were significantly genetically isolated from each other, with private mtDNA haplotypes and microsatellite alleles, indicating limited gene flow among populations, strong effects of genetic drift due to small Ne, or a combination of both. The Mantel test of microsatellites revealed a significant correlation (r = 0.414, P = 0.04) between genetic and geographic distances for pairwise comparisons among the ten populations, while that of mtDNA showed a lack of correlation. Given the shared identical mtDNA haplotype and similar microsatellite allelic distributions between Chiak and Hongcheon populations, we suggest that the restored (introduced) Chiak population would be inferred to be genetically originated from Hongcheon population. Phylogenetic relationships among Northeast Asian populations showed that South Korean lineages have more recently diverged from China (Yellow River), than between North Korea and Russia. Although the phylogenetic relationship would be expected to be associated with geography, South-North Korea and China populations with a similar latitude was more phylogenetically closely related. These findings may suggest a possible scenario for the historical movements of B. lenok tsinlingensis in Northeast Asia during Last Glacial Maximum (LGM). It would be supported by the line of evidence that most lenok populations migrated to southward from Northern Asia such as Russia and Mongolia during LGM because the Korean Peninsula was landlocked as inland epoch and functioned as a southern shelter with Yellow River. For this reason, the Korean Peninsula is suggested to be an important geographical region for better understanding phylogenetic relationships and evolutionary histories of B. lenok tsinlingensis across the Northeast Asia. Despite large efforts made to develop several restoration programs in South Korea for B. lenok tsinlingensis, it is still unknown whether these past restoration efforts were successful or fruitless, mainly because of little attention paid to post-restoration monitoring research. Hence, there was a lack of their published official records. In the future, conservation and restoration projects of the Korean lenok populations should consider the genetic data for a better understanding of their ecological and evolutionary trajectories. And finally, we hope that our findings here can help inform on the future effective conservation and restoration plans for B. lenok tsinlingensis populatio ns in South Korea.

Seagrasses, sea flowering plants, comprise approximately 60 species globally and are often called ‘ecosystem engineers’ because they create their own habitats by modifying the surrounding environments, which provide coastal zones with a number of crucial ecosystem services. Zostera marina (the common name ‘eelgrass’) is one of the seagrass beds-forming species distributed widely in northern hemisphere including the Korean coast, which plays a pivotal role in ecosystem as a primary producer and a nursery habitat or refuge for other marine organisms. However, due to global climate change and anthropogenic activities such as reclamation and dredging, there has recently been a drastic decline in population sizes of Z. marina in Korea. In order to develop effective conservation and restoration management programs of Z. marina populations, it would be helpful to consider all biological aspects of this species such as genetic characteristics as well as ecological and physiological features. This study first provides information on genetic diversity and genetic structure of Jeju Island and Namhae populations of Z. marina, which will contribute to the establishment of appropriate conservation and restoration management plans for future persistence of this species. Using six microsatellite markers, we investigated the level of genetic diversity and genetic structure among 10 geographic populations of Z. marina inhabiting Jeju Island (Hamdeok, Tokki-seom, Sungsan, Woljeong, Ojo) and Namhae (Gamak bay, Jindong bay, Nampo, Anggang bay, Geoje) on the southern coast of Korea. The level of genetic diversity within Jeju populations (mean allelic richness [AR]: 1.57 ~ 3.09) was found to be significantly lower than Namhae populations (AR: 3.09 ~ 4.29) (Mann-Whitney U-test, P < 0.05). These findings suggest that effective population sizes (Ne) of Jeju populations are generally smaller than those of Namehae populations. Within Jeju Island, Hamdeok population had the smallest population size (coverage: 138 m2) and the lowest genetic diversity (AR: 1.57), while Ojo population had the largest population size (coverage: 275,736 m2) and the greatest level of genetic diversity (AR: 3.09). Hamdeok population showed evidence of genetic bottleneck. These results again suggest that Ne of Jeju populations is generally low (except Ojo population). Among Jeju populations, all pair-wise comparisons of FST values (i.e., degree of genetic differentiation) were highly significant (FST = 0.0612 ~ 0.7168, P < 0.001) despite Jeju populations that were geographically closely located, indicating that these local populations are genetically divergent, probably due to a lack of gene flow among the populations. The observed strong population structure was substantiated by evidence that five genetic clusters are most likely, based on population assignment test (STRUCTURE). The Mantel test showed a positive relationship between genetic distance (FST) and geographic distance (km) across all the populations sampled (R2 = 0.4118, P < 0.05), suggesting that our data follow Isolation By Distance (IBD) model. Woljeong population revealed the highest level of FST values compared to other populations within Jeju Island in IBD. STRUCTURE and factorial correspondence analysis (FCA) further showed that some Woljeong individuals included genotypes of Namhae populations. Population size of Woljeong (coverage: 310m2) was approximately 50 % smaller than that of Sungsan (coverage: 841m2); however, extent of its genetic diversity (AR: 2.39) was even higher than that of Sungsan population (AR: 1.77). We speculated that Woljeong population underwent a transplantation from Namhae populations with relatively higher level of genetic diversity. FST values within Namhae populations were relatively lower (compared to within Jeju Island) despite the populations that were geographically more distant. It means that level of gene flow is higher among Namhae populations than among Jeju populations. Z. marina is known to have different life histories by water depth. In subtidal zone (deep water depth) populations predominantly undertake sexual reproduction through seeds such as annual life history, whereas those of intertidal zone (shallow water depth) undertake both sexual and asexual reproductions through horizontal rhizomes i.e., perennial life history. STRUCTURE analysis showed no clear differences between shallow and deep populations at Namhae, but some FST values were statistically significantly different despite their low values. For Geoje population sampled in 2005, intertidal and subtidal populations were not significantly different (FST = 0.0045, P = 0.033), but these populations sampled in 2015 showed a significant difference (FST = 0.0328, P < 0.001). It means that genetic structure of Geoje has been changed over the 10 year period between shallow and deep populations. Overall, the Jeju and Namehae populations analyzed in the current study have relatively low levels of genetic diversity and distinct genetic compositions, which warns the message that this ecologically important species should be conserved separately in the local populations and with high priority. We propose that future conservation and restoration plans for seagrasses should consider genetic characteristics particularly because a close relationship between genetic diversity and ecological performance in marine species has been well documented.