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Methodologies for Cryopreservation of Mammalian Germline Cells and Tissues
Polash Chandra Karmakar,Sang-Eun Jung,Buom-Yong Ryu 한국동물생명공학회(구 한국동물번식학회) 2017 Reproductive & developmental biology Vol.41 No.2
Until today, success in germline cells and tissue cryopreservation is limited mainly due to the poor understanding of the complex physiological processes can lead to cell damage during cryopreservation. Germline cells, from both male and female, have unique ability to differentiate into one or more cell lines and thus it becomes a crucial point to store them in subzero temperature with the minimal damage of their functional properties and maximum recovery of unchanged and viable cells when thawed. In the past three decades, a vast research has been performed using various different animal models which in fact have led to development of new methodologies and optimization of older one. However, successful use of animal model has provided the opportunity in research with human germline cells and tissues preservation, but not in all the cases. Therefore, the use of new cryo-protective chemicals and modified protocols have been often found in different groups of researchers based on the types, physical structures, utility and animal species of the specimens to be cryopreserved. This review discusses about the basics of different types of cryopreservation methodologies and commonly used optimized protocols and cryoprotectants for germline cells and tissues preservation.
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Methodologies for Cryopreservation of Mammalian Germline Cells and Tissues
Karmakar, Polash Chandra,Jung, Sang-Eun,Ryu, Buom-Yong The Korean Society of Animal Reproduction 2017 Reproductive & developmental biology Vol.41 No.2
Until today, success in germline cells and tissue cryopreservation is limited mainly due to the poor understanding of the complex physiological processes can lead to cell damage during cryopreservation. Germline cells, from both male and female, have unique ability to differentiate into one or more cell lines and thus it becomes a crucial point to store them in subzero temperature with the minimal damage of their functional properties and maximum recovery of unchanged and viable cells when thawed. In the past three decades, a vast research has been performed using various different animal models which in fact have led to development of new methodologies and optimization of older one. However, successful use of animal model has provided the opportunity in research with human germline cells and tissues preservation, but not in all the cases. Therefore, the use of new cryo-protective chemicals and modified protocols have been often found in different groups of researchers based on the types, physical structures, utility and animal species of the specimens to be cryopreserved. This review discusses about the basics of different types of cryopreservation methodologies and commonly used optimized protocols and cryoprotectants for germline cells and tissues preservation.
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Enrichment and In Vitro Culture of Spermatogonial Stem Cells from Pre-Pubertal Monkey Testes
김용희,강현구,김방진,정상은,Polash C. Karmakar,김석만,황성수,류범용 한국조직공학과 재생의학회 2017 조직공학과 재생의학 Vol.14 No.5
Spermatogonial stem cells (SSCs) are essential for spermatogenesis throughout the lifespan of the male. However, the rarity of SSCs has raised the need for an efficient selection method, but little is known about culture conditions that stimulate monkey SSC proliferation in vitro. In this study, we report the development of effective enrichment techniques and in vitro culturing of germ cells from pre-pubertal monkey testes. Testis cells were analyzed by fluorescence-activated cell sorting techniques and were transplanted into the testes of nude mice to characterize SSCs. Thy- 1-positive cells showed a higher number of colonies than the unselected control after xenotransplantation. Extensive colonization of monkey cells in the mouse testes indicated the presence of highly enriched populations of SSCs in the Thy- 1-positive sorted cells. Furthermore, monkey testis cells were enriched by differential plating using extracellular matrix, laminin, and gelatin, and then cultured under various conditions. Isolation of monkey testicular germ cells by differential plating increased germ cell purity by 2.7-fold, following the combinational isolation method using gelatin and laminin. These enriched germ cells actively proliferated under culture conditions involving StemPro medium supplemented with bFGF, GDNF, LIF, and EGF at 37 C. These results suggest that the enrichment and in vitro culture method proposed in the present study for harvesting a large number of functionally active monkey SSCs can be applied as the basis for efficient in vitro expansion of human SSCs.
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Myeong-Geun Oh,Yong-Hee Kim,Polash Chandra Karmakar,Sang-Eun Jung,Seok-Man Kim,Ju-Hee Jin,Buom-Yong Ryu 한국동물생명공학회(구 한국동물번식학회) 2017 Reproductive & Developmental Biology(Supplement) Vol.41 No.2
Spermatogonial stem cells (SSCs) are the basis of spermatogenesis in male. Among the testicular germ cells, SSCs are considered as an important population because of their capability to multiply in numbers which is also known as self-renewal, and to differentiate into spermatocytes by subsequent meiotic processes finalized with the production of spermatozoa. As SSCs are present in a very small proportion among the total testis cells, in vitro proliferation of SSCs in undifferentiated state is very important in the field of experiments related to germ cell culture. The objective of the study was to establish an effective feeder cell for murine germ cell development in vitro. We used three types of feeder cells to grow murine germ cells, such as STO, tissue specific feeder layer, and C166 feeder cells and green fluorescence protein (GFP) rat SSCs were used to grow on them. A continuous culture was performed for each feeder groups, but C166 feeder cells found unable to proliferate germ cells even after several weeks. We observed a comparatively better proliferation rate of rat germ cells grown on tissue specific feeder layer where cultured germ cell colony and cell count were higher than STO feeder. Immunocytochemistry of undifferentiated SSC marker showed significantly higher expression for germ cell cultured on tissue specific feeder layer. Similarly, mRNA level of undifferentiated spermatogonia were found higher in case of tissue specific feeder layer and STO, although the expression level of differentiated spermatogonia was similar. Finally, the functional characteristics of SSCs were studied by cultured germ cell transplantation into recipient mice. We used donor rat germ cells cultured on both feeder groups for 4 months, where SSCs from tissue specific feeder layer showed higher functional and stemness activity. Transplantation of SSCs cultured for 8 months from tissue specific feeder layer group were performed to recipient rat and we could produce GFP offspring from those recipients. Thus the experiment conclude that tissue specific cells could be an effective feeder layer for murine SSCs culture based on their survivability and proliferation along with retaining the perfect functional characteristics and stemness properties.
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Rahman, Md Saidur,Kwon, Woo-Sung,Ryu, Do-Yeal,Khatun, Amena,Karmakar, Polash Chandra,Ryu, Buom-Yong,Pang, Myung-Geol American Chemical Society 2018 JOURNAL OF PROTEOME RESEARCH Vol.17 No.1
<P>Studies regarding bisphenol A (BPA) exposure and male (in)fertility have conventionally focused on modifications in ejaculated spermatozoa function from exposed individuals. However, mammalian spermatozoa are incapable of fertilization prior to achieving capacitation, the penultimate step in maturation. Therefore, it is necessary to investigate BPA-induced changes in capacitated spermatozoa and assess the consequences on subsequent fertilization. Here, we demonstrate the effect of gestational BPA exposure (50 μg/kg bw/day, 5 mg/kg bw/day, and 50 mg/kg bw/day) on the functions, biochemical properties, and proteomic profiles of F1 capacitated spermatozoa from adult mice. The data showed that high concentrations of BPA inhibited motility, motion kinematics, and capacitation of spermatozoa, perhaps because of increased lipid peroxidation and protein tyrosine nitration, and decreased intracellular ATP levels and protein kinase-A activity in spermatozoa. We also found that BPA compromised the rates of fertilization and early embryonic development. Differentially expressed proteins identified between BPA-exposed and control groups play a critical role in energy metabolism, stress responses, and fertility. Protein function abnormalities were responsible for the development of several diseases according to bioinformatics analysis. On the basis of these results, gestational exposure to BPA may alter capacitated spermatozoa function and the proteomic profile, ultimately affecting their fertility potential.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jprobs/2018/jprobs.2018.17.issue-1/acs.jproteome.7b00668/production/images/medium/pr-2017-00668k_0005.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/pr7b00668'>ACS Electronic Supporting Info</A></P>
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Prognostic role of EGR1 in breast cancer: a systematic review
( Subbroto Kumar Saha ),( S. M. Riazul Islam ),( Tripti Saha ),( Afsana Nishat ),( Polash Kumar Biswas ),( Minchan Gil ),( Lewis Nkenyereye ),( Shaker El-sappagh ),( Saiful Islam ),( Ssang-goo Cho ) 생화학분자생물학회 2021 BMB Reports Vol.54 No.10
EGR1 (early growth response 1) is dysregulated in many cancers and exhibits both tumor suppressor and promoter activities, making it an appealing target for cancer therapy. Here, we used a systematic multi-omics analysis to review the expression of EGR1 and its role in regulating clinical outcomes in breast cancer (BC). EGR1 expression, its promoter methylation, and protein expression pattern were assessed using various publicly available tools. COSMIC-based somatic mutations and cBioPortal-based copy number alterations were analyzed, and the prognostic roles of EGR1 in BC were determined using Prognoscan and Kaplan-Meier Plotter. We also used bc-GenEx-Miner to investigate the EGR1 co-expression profile. EGR1 was more often downregulated in BC tissues than in normal breast tissue, and its knockdown was positively correlated with poor survival. Low EGR1 expression levels were also associated with increased risk of ER+, PR+, and HER2- BCs. High positive correlations were observed among EGR1, DUSP1, FOS, FOSB, CYR61, and JUN mRNA expression in BC tissue. This systematic review suggested that EGR1 expression may serve as a prognostic marker for BC patients and that clinicopathological parameters influence its prognostic utility. In addition to EGR1, DUSP1, FOS, FOSB, CYR61, and JUN can jointly be considered prognostic indicators for BC. [BMB Reports 2021; 54(10): 497-504]
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The extract of natural plant promotes the proliferation of murine spermatogonial stem cells
Hye-Ryeon Kang,Yong-Hee Kim,Dong-Gu Lee,Ki-Jung Kim,Polash Chandra Karmakar,Hyun-Gu Kang,Sang-Eun Jung,Myeong-Geun Oh,Yeon-Jim Cho,Yu-Ri Choi,Chan Kyu Han,Sanghyung Lee,Buom-Yong Ryu 한국발생생물학회 2015 한국발생생물학회 학술발표대회 Vol.2015 No.9
The aim of this study was to enhance the proliferation efficiency of spermatogonial stem cells (SSCs). In order to improve the proliferation efficiency, we investigated new factors that promote the proliferation of SSCs using in vitro culture method with natural plant extracts. Germ cell populations containing SSCs were collected 6- to 8-days-old from C57BL/6-TG-EGFP (C57GFP) mice and SSCs were isolated from the collected cells via magnetic-activated cell sorting (MACS). Since then, SSCs were cultured for a week with culture medium containing natural plant extracts at concentration of 0.1, 1, and 10 μg/mL. After a week of culture, we looked for an increase, especially a dose-dependent increase, in the number of cells compared to that of the control group. A dose-dependent increase, in the number of cells was observed in the Petasides japonicus-treated groups. Furthermore, we carried out repeated experiment that is process consisting of selection and additional segmentation to explore new factors for activating SSCs at the molecular level. As a results, Petasides japonicus butanol fraction significantly increased the proliferation rate of SSCs in a dose-dependent manner among Petasides japonicus fraction samples. We identified normal expression level of PLZF in SSCs cultured with plant extracts using immunocytochemistry method. Furthermore, we also carried out qRT-PCR and identified normal expression level of Lhx1 and GFRα1. The finding of this study could contribute to improvement of proliferation and activation for SSCs, using culture method with natural plant extracts.
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Effect of Necrostatin-1 on Cryopreservation of Mouse Spermatogonial Stem Cells
Sang-Eun Jung,Yong-Hee Kim,Yeon-Jin Cho,Polash Chandra Karmakar,Myeong-geun Oh,Yu-ri Choi,Buom-Yong Ryu 한국수정란이식학회 2016 한국수정란이식학회 학술대회 Vol.2016 No.10
Spermatogonial stem cells (SCCs) is foundation for spermatogenesis throughout male adult life because they have ability of self-renewal and differentiation into spermatozoa. Storage of such SSCs is very important to study on male reproduction, which would contribute human male infertility to be treated. However, during cryopreservation, the most cells are damaged by cryoinjury such as apoptosis, necrosis, osmotic stress, oxidative stress and so on. For the reason, in cryopreservation technique, targeting purpose is what cells are stored stably without cryoinjury. The purpose of this study was to develop the cryoprotectant for decrease in cryoinjury of SSCs by using melatonin and necrostatin-1 as additive cryoprotectant. The SSCs with melatonin or necrostatin-1 was frozen for 1 month, and then thawed to evaluate survival, recovery and proliferation rate. The result showed that necrostatin-1 50 mM was significantly greater than DMSO control. Furthermore, we conducted the characterization of cryo-thawed SSCs with necrostatin-1 50 mM to confirm whether the SSCs could maintain the undifferentiated state. As a result, the normal expression of each marker, which is PLZF, GFRa1 and VASA, was observed except for C-kit, meaning that the cells could maintain the undifferentiated state regardless of cryopreservation. Therefore, the result indicates that the cryo-thawed SSCs have ability of proliferation and self-renewal. In conclusion, our finding verifies that cryopreservation of SSC with necrostatin-1 50 mM could be helpful to preserve the SSCs stably, contributing to various studies on male reproduction and infertility treatment
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