Mol. Cells 2014; 37(6): 473-479
Published online May 23, 2014
https://doi.org/10.14348/molcells.2014.0080
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: knko@kku.ac.kr
Spermatogonial stem cells (SSCs, also called germline stem cells) are self-renewing unipotent stem cells that produce differentiating germ cells in the testis. SSCs can be isolated from the testis and cultured
Keywords cell culture, feeder-free, matrigel, spermatogonial stem cells
Spermatogonial stem cells (SSCs), also called germline stem cells, are unipotent precursor cells that self-renew and contribute to spermatogenesis in the testis. Although SSCs represent an extremely rare population (0.02-0.03%) in the testis (Tegelenbosch and de Rooij, 1993), they can be isolated and propagated
SSCs require a specific culture system for successful longterm expansion
Matrigel, extracted from the Engelbreth-Holm-Swarm mouse tumors, contains several extracellular matrix (ECM) molecules, such as laminin, collagen, entactin, heparan sulfate proteoglycan, and also growth factors, such as fibroblast growth factor (FGF), EGF, insulin-like growth factor 1 (IGF-1), transforming growth factor beta (TGF-β), and platelet-derived growth factor (PDGF) (Braam et al., 2008; Vukicevic et al., 1992). Matrigel is widely used as the feeder-free substrate to mimic the ECM for cell culture, presumably by replicating cell-ECM interactions. Matrigel has been shown to provide an optimal microenvironment for stem cell culture, especially for embryonic stem cells (ESCs), because of its ability to maintain self-renewality and pluripotency of ESCs (Mallon et al., 2006). In the present study, we evaluated the ability of Matrigel to support the attachment of SSCs and their long-term maintenance
We found that feeder-free cultured SSCs proliferated for at least 5 months at a rate comparable to that of SSCs cultured on MEFs. During this time, SSCs retained their cellular properties and functionality. Our feeder-free culture systems have a potential to enable studies of regulatory mechanisms that determine the SSC fate in an efficient and cost-effective way.
SSCs were established from Oct4-GFP and Oct4-GFP/LacZ transgenic mice (C57BL/6 background) as previously described (Ko et al., 2009; 2010; 2012). After a two-step digestion of testicular tubules, testicular cells were plated onto gelatin-coated culture dishes (2 × 105 cells/3.8 cm2) with SSC culture medium. SSC colonies were observed under the microscope within 7 days. SSC colonies were collected by gentle pipetting and replated on mitomycin C-treated MEFs for expansion (Ko et al., 2009). SSCs were maintained on MEFs or feeder-free (Matrigel- coated) plates and passaged every 5 days. Cell numbers were counted at each passage; cells were replated (5 × 105 cells/well) in 12-well plates. Experiments were conducted under protocols approved by the Konkuk University Animal Care and Use Committee.
SSC medium was composed of StemPro-34 SFM (Invitrogen) with the following supplements: StemPro supplement (Invitrogen), 1× N2 supplement (Invitrogen), 6 mg/ml d-(+)- glucose (Invitrogen), 30 mg/ml pyruvic acid (Invitrogen), 1 μl/ml DL-lactic acid (Sigma), 5 mg/ml bovine serum albumin (BSA; Invitrogen), 1% fetal bovine serum (Invitrogen), 2 mM Lglutamine (Invitrogen), 50 μM β-mercaptoethanol (Invitrogen), 1 × penicillin/streptomycin (Welgene), 1× minimal essential medium (MEM) non-essential amino acids (Invitrogen), 1× MEM vitamins (Invitrogen), 30 ng/ml β-estradiol (Sigma), 60 ng/ml progesterone (Sigma), 20 ng/ml human EGF (Peprotech), 20 ng/ml human bFGF (Peprotech), 20 ng/ml human GDNF (Peprotech), and 103 U/ml murine leukemia inhibitory factor (Prospec).
Culture plates were coated with ECM, Matrigel (BD Biosciences) or laminin (Sigma) by the following procedure. A Matrigel bottle was thawed on ice in a 4°C refrigerator overnight until Matrigel liquified. Matrigel was divided into 300 μl aliquots and stored at -20°C until use. For preparation of Matrigel-coated plates, working Matrigel solution was prepared by diluting 300 μl of Matrigel with 29 ml of DMEM/F12 medium (Invitrogen) and thorough mixing. This solution was added to 12-well plates (0.5 ml per well) or 6-well plates (1 ml per well) to cover the whole surface of the wells. The plates were allowed to sit for 1 h at room temperature or overnight at 4°C. Excess Matrigel solution was then removed, and the plates were washed once with DMEM/F12. For laminin coating, laminin stock solution (1 mg/ml) was divided into 50 μl aliquots and stored at -20°C. Laminin working solutions (20 μg/ml) were prepared by diluting the stock solution with Dulbecco's phosphate-buffered saline (DPBS), and added to 12-well plates (0.5 ml per well) or 6-well plates (1 ml per well). Plates were incubated with laminin solution for at least 2 h in a 37°C in a cell culture incubator, then excess laminin solution was removed and the plates were washed twice with DPBS.
Total RNA was isolated by using the miRNeasy Mini Kit according to the manufacturer’s instructions (QIAGEN). Total RNA (500 ng) was reverse-transcribed by using the Omniscript RT Kit (QIAGEN) in a total volume of 20 μl. PCR analysis was performed with gene-specific primers (Supplementary Table 1) and Takara Ex Taq DNA polymerase (Takara) according to the manufacturer’s instructions. The PCR conditions were as follows: 32 cycles at 94°C for 30 s, 50-65°C for 30 s, and 72°C for 30 s. The RT-PCR products were analyzed by electrophoresis in 1% agarose gels.
SSCs were resuspended in ice-cold DPBS containing 0.5% BSA at 1 × 106 cells/ml, and incubated for 20 min on ice with the following antibodies: CD326 (EpCAM), CD49f (Intergrin α6), fluorescein isothiocyanate (FITC)-conjugated CD29 (Intergrin β1) (all from BD Biosciences), or phycoerythrin-conjugated CD117 (c-kit) (Biolegend). Cells were then washed with 0.5% BSA in PBS and incubated with secondary antibodies: antimouse IgM-FITC (Sigma) for CD326 (EpCAM), anti-rat IgGFITC (BD Biosciences) for CD49f (Intergrin α6). Analysis was performed by flow cytometry (BD Biosciences) using the CellQuest software (BD Biosciences).
Cells were washed with PBS, and genomic DNA was isolated using the Total DNA Extraction kit (iNtRON) according to the manufacturer’s protocol. Genomic DNA was treated with EpiTech Bisulfite (QIAGEN) according to the manufacturer’s recommendation, and used for PCR amplification. The PCR products were subcloned using the PCR Cloning kit (QIAGEN). Primers for bisulfite sequencing are listed in Supplementary Table 1.
Transplantation was performed as described (Ko et al., 2009). Six-week-old C57BL/6 male mice were treated with 40 mg/kg busulfan by intraperitoneal injection and used as recipients 1 month after busulfan treatment. SSC medium (approximately 10 μl) containing 5 × 105 cells and 0.04% trypan blue was injected with a micropipette (40-60 μm diameter tips) into the seminiferous tubules of the testes of recipient mice through the efferent duct (which connects the testis to the epididymis) (Ko et al., 2012). The mice were sacrificed 2 months later, and their testicular tubules were stained for LacZ to reveal positive cells corresponding to colonization by transplanted SSCs.
SSCs colonies have a typical grape-like morphology when cultured on mitomycin C-treated MEFs (Figs. 1A and 1B). For expansion of the SSCs cultured on feeder cells, feeders have to be prepared for every subculture. This approach has considerable drawbacks, as it involves several tedious and timeconsuming procedures, including culturing MEFs and their mitotic inactivation with mitomycin C. Therefore, a feeder-free culture system would allow maintaining and propagating SSCs in a labor- and cost-effective manner.
SSCs in the testis are located and maintained on the basal membrane of testicular tubules. This implies that the
To examine the expandability of SSCs cultured on Matrigel, we replated them at 5 × 105 cells per well of a 12 well plate every 5 days. The proliferation ability was maintained for more than 100 days at rates of approximately 2.5-fold per 5 days, which was similar to proliferation rates in the feeder culture system (Figs. 1E and 1F). This suggests that Matrigel can support unlimited
To characterize SSCs cultured in Matrigel-based feeder-free conditions, we performed RT-PCR and FACS analysis to examine SSC-specific gene expression at the mRNA (MEF and testis samples used as negative control and positive control, respectively) and surface protein levels, respectively. As shown in Fig. 2A, SSCs cultured under feeder-free conditions expressed the SSC marker genes
To investigate the epigenetic stability of SSCs cultured in the feeder-free system, we conducted bisulfite DNA sequencing analysis to determine the genomic imprinting pattern in these SSCs. DNA methylation patterns of the differentially methylated regions (DMRs) of the paternally imprinted gene
To determine the stem cell activity of cultured SSCs, we performed the spermatogonial transplantation assay. Because there is no clear functional
SSCs generally require the basement membrane to maintain their stem cell properties both
Several previous attempts have been made to culture SSCs under feeder-free conditions using laminin (Kanatsu-Shinohara et al., 2005; 2011), but in this case new SSC culture medium components were required. However, our feeder-free culture system uses the same SSC medium as the feeder culture systems, so no new components need to be prepared. Furthermore, we found an obvious difference in the SSC proliferation rate between laminin and Matrigel (Supplementary Fig. 1). Although it was not significantly different during the first 3 passages, SSC proliferation on laminin was dramatically reduced after 4 passages. This suggests that Matrigel is more suitable for maintaining proliferating SSC cultures
It should be noted that efficient propagation of SSCs on Matrigel requires more than 5 × 105 cells per well of a 12 well plate. Our feeder-free culture is cell number-dependent with the optimal cell density of approximately 1.3 × 105 cells/cm2. It is possible that the cell number-dependent growth of SSCs under feeder-free conditions may be related to the action of survivalenhancing autocrine factors, which is well described in ESCs (Mittal and Voldman, 2011).
We chose Matrigel as a matrix in our feeder-free culture for SSCs. Matrigel is the manufacturer’s trademark for ECM extracted from the Engelbreth-Holm-Swarm tumors (Vukicevic et al., 1992). In our initial experiments on feeder-free SSC culture, we used standard Matrigel that contains a mixture of ECM molecules and growth factors, such as EGF, IGF-1, TGF-β and PDGF. Later, we switched to growth factor?reduced (GFR) Matrigel for use in the feeder-free cultures. We found no difference between standard Matrigel and GFR-Matrigel in their ability to support SSC proliferation (Supplementary Fig. 3). This suggests that the growth factors in standard Matrigel may not influence SSC proliferation under feeder-free culture conditions, which was also confirmed by the results that adding IGF-1, TGF-β, and PDGF did not support proliferation of SSCs on gelatin-coated dishes. Thus, a combination of the major ECM components, such as laminin, collagen, entactin, and heparan sulfate proteoglycan, likely plays a role in creating proper basement membrane-like environment for SSCs in our feederfree culture.
Our expression analysis of genes for intracellular and cell surface SSCs markers revealed no differences between our feeder-free culture system and a feeder culture. DNA imprinting patterns of SSCs were also preserved in the feeder-free culture: the methylation status was androgenetic in both culture types. Furthermore, we found that SSCs transplanted to the male mouse testicular tubes were able to colonize the recipient testis. Therefore, the feeder-free culture system can maintain the cellular and molecular phenotypes as well as functionality of SSCs.
Our feeder-free culture system for expansion of SSCs is simple and efficient, because it uses the same SSC medium as the feeder cultures. Moreover, in comparison with the lamininbased culture, our Matrigel-based feeder-free culture system is superior as it allows long-term cell proliferation, whereas laminin cannot support SSC proliferation after 3 passages.
In this context, the Matrigel-based culture system provides time- and cost-effective unified approaches for feeder-free expansion of SSCs. Moreover, as it excludes variability caused by the presence of feeder cells, this system should be suitable for more definitive experiments to study the molecular mechanisms regulating SSCs, which is important in basic reproductive and stem cell biology, and also for therapeutic applications of germ cells in infertility treatment.
In this study, we developed a novel feeder-free culture system to support long-term maintenance of SSCs. We have demonstrated that our culture system preserves SSC properties properties at the molecular and cellular levels and their functionality. The advantage of our feeder-free culture system is that it provides a time- and cost-effective method to maintain SSCs, because it does not require feeders and uses the same medium as feeder cultures. Thus, our culture protocol will make culturing SSCs simple and reproducible, facilitate scale-up, and allow definitive experiments to evaluate individual factors affecting SSC stemness, which is important both in basic and applied reproductive biology.
Mol. Cells 2014; 37(6): 473-479
Published online June 30, 2014 https://doi.org/10.14348/molcells.2014.0080
Copyright © The Korean Society for Molecular and Cellular Biology.
Na Young Choi1,2, Yo Seph Park1,2, Jae-Sung Ryu2, Hye Jeong Lee1,2, Marcos J. Ara?zo-Bravo3,4, Kisung Ko5, Dong Wook Han1,2, Hans R. Sch?ler6,7, and Kinarm Ko1,2,8,*
1Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul 143-701, Korea, 2Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 143-701, Korea, 3Group of Computational Biology and Bioinformatics, Biodonostia Health Research Institute, San Sebasti?n, Spain, 4IKERBASQUE, Basque Foundation for Science, Bilbao, Spain, 5Department of Medicine, Medical Research Institute, College of Medicine, Chung-Ang University, Seoul 156-756, Korea, 6Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, M?nster 48149, Germany, 7University of M?nster, Medical Faculty, 48149 M?nster, Germany, 8Research Institute of Medical Science, Konkuk University, Seoul 143-701, Korea
Correspondence to:*Correspondence: knko@kku.ac.kr
Spermatogonial stem cells (SSCs, also called germline stem cells) are self-renewing unipotent stem cells that produce differentiating germ cells in the testis. SSCs can be isolated from the testis and cultured
Keywords: cell culture, feeder-free, matrigel, spermatogonial stem cells
Spermatogonial stem cells (SSCs), also called germline stem cells, are unipotent precursor cells that self-renew and contribute to spermatogenesis in the testis. Although SSCs represent an extremely rare population (0.02-0.03%) in the testis (Tegelenbosch and de Rooij, 1993), they can be isolated and propagated
SSCs require a specific culture system for successful longterm expansion
Matrigel, extracted from the Engelbreth-Holm-Swarm mouse tumors, contains several extracellular matrix (ECM) molecules, such as laminin, collagen, entactin, heparan sulfate proteoglycan, and also growth factors, such as fibroblast growth factor (FGF), EGF, insulin-like growth factor 1 (IGF-1), transforming growth factor beta (TGF-β), and platelet-derived growth factor (PDGF) (Braam et al., 2008; Vukicevic et al., 1992). Matrigel is widely used as the feeder-free substrate to mimic the ECM for cell culture, presumably by replicating cell-ECM interactions. Matrigel has been shown to provide an optimal microenvironment for stem cell culture, especially for embryonic stem cells (ESCs), because of its ability to maintain self-renewality and pluripotency of ESCs (Mallon et al., 2006). In the present study, we evaluated the ability of Matrigel to support the attachment of SSCs and their long-term maintenance
We found that feeder-free cultured SSCs proliferated for at least 5 months at a rate comparable to that of SSCs cultured on MEFs. During this time, SSCs retained their cellular properties and functionality. Our feeder-free culture systems have a potential to enable studies of regulatory mechanisms that determine the SSC fate in an efficient and cost-effective way.
SSCs were established from Oct4-GFP and Oct4-GFP/LacZ transgenic mice (C57BL/6 background) as previously described (Ko et al., 2009; 2010; 2012). After a two-step digestion of testicular tubules, testicular cells were plated onto gelatin-coated culture dishes (2 × 105 cells/3.8 cm2) with SSC culture medium. SSC colonies were observed under the microscope within 7 days. SSC colonies were collected by gentle pipetting and replated on mitomycin C-treated MEFs for expansion (Ko et al., 2009). SSCs were maintained on MEFs or feeder-free (Matrigel- coated) plates and passaged every 5 days. Cell numbers were counted at each passage; cells were replated (5 × 105 cells/well) in 12-well plates. Experiments were conducted under protocols approved by the Konkuk University Animal Care and Use Committee.
SSC medium was composed of StemPro-34 SFM (Invitrogen) with the following supplements: StemPro supplement (Invitrogen), 1× N2 supplement (Invitrogen), 6 mg/ml d-(+)- glucose (Invitrogen), 30 mg/ml pyruvic acid (Invitrogen), 1 μl/ml DL-lactic acid (Sigma), 5 mg/ml bovine serum albumin (BSA; Invitrogen), 1% fetal bovine serum (Invitrogen), 2 mM Lglutamine (Invitrogen), 50 μM β-mercaptoethanol (Invitrogen), 1 × penicillin/streptomycin (Welgene), 1× minimal essential medium (MEM) non-essential amino acids (Invitrogen), 1× MEM vitamins (Invitrogen), 30 ng/ml β-estradiol (Sigma), 60 ng/ml progesterone (Sigma), 20 ng/ml human EGF (Peprotech), 20 ng/ml human bFGF (Peprotech), 20 ng/ml human GDNF (Peprotech), and 103 U/ml murine leukemia inhibitory factor (Prospec).
Culture plates were coated with ECM, Matrigel (BD Biosciences) or laminin (Sigma) by the following procedure. A Matrigel bottle was thawed on ice in a 4°C refrigerator overnight until Matrigel liquified. Matrigel was divided into 300 μl aliquots and stored at -20°C until use. For preparation of Matrigel-coated plates, working Matrigel solution was prepared by diluting 300 μl of Matrigel with 29 ml of DMEM/F12 medium (Invitrogen) and thorough mixing. This solution was added to 12-well plates (0.5 ml per well) or 6-well plates (1 ml per well) to cover the whole surface of the wells. The plates were allowed to sit for 1 h at room temperature or overnight at 4°C. Excess Matrigel solution was then removed, and the plates were washed once with DMEM/F12. For laminin coating, laminin stock solution (1 mg/ml) was divided into 50 μl aliquots and stored at -20°C. Laminin working solutions (20 μg/ml) were prepared by diluting the stock solution with Dulbecco's phosphate-buffered saline (DPBS), and added to 12-well plates (0.5 ml per well) or 6-well plates (1 ml per well). Plates were incubated with laminin solution for at least 2 h in a 37°C in a cell culture incubator, then excess laminin solution was removed and the plates were washed twice with DPBS.
Total RNA was isolated by using the miRNeasy Mini Kit according to the manufacturer’s instructions (QIAGEN). Total RNA (500 ng) was reverse-transcribed by using the Omniscript RT Kit (QIAGEN) in a total volume of 20 μl. PCR analysis was performed with gene-specific primers (Supplementary Table 1) and Takara Ex Taq DNA polymerase (Takara) according to the manufacturer’s instructions. The PCR conditions were as follows: 32 cycles at 94°C for 30 s, 50-65°C for 30 s, and 72°C for 30 s. The RT-PCR products were analyzed by electrophoresis in 1% agarose gels.
SSCs were resuspended in ice-cold DPBS containing 0.5% BSA at 1 × 106 cells/ml, and incubated for 20 min on ice with the following antibodies: CD326 (EpCAM), CD49f (Intergrin α6), fluorescein isothiocyanate (FITC)-conjugated CD29 (Intergrin β1) (all from BD Biosciences), or phycoerythrin-conjugated CD117 (c-kit) (Biolegend). Cells were then washed with 0.5% BSA in PBS and incubated with secondary antibodies: antimouse IgM-FITC (Sigma) for CD326 (EpCAM), anti-rat IgGFITC (BD Biosciences) for CD49f (Intergrin α6). Analysis was performed by flow cytometry (BD Biosciences) using the CellQuest software (BD Biosciences).
Cells were washed with PBS, and genomic DNA was isolated using the Total DNA Extraction kit (iNtRON) according to the manufacturer’s protocol. Genomic DNA was treated with EpiTech Bisulfite (QIAGEN) according to the manufacturer’s recommendation, and used for PCR amplification. The PCR products were subcloned using the PCR Cloning kit (QIAGEN). Primers for bisulfite sequencing are listed in Supplementary Table 1.
Transplantation was performed as described (Ko et al., 2009). Six-week-old C57BL/6 male mice were treated with 40 mg/kg busulfan by intraperitoneal injection and used as recipients 1 month after busulfan treatment. SSC medium (approximately 10 μl) containing 5 × 105 cells and 0.04% trypan blue was injected with a micropipette (40-60 μm diameter tips) into the seminiferous tubules of the testes of recipient mice through the efferent duct (which connects the testis to the epididymis) (Ko et al., 2012). The mice were sacrificed 2 months later, and their testicular tubules were stained for LacZ to reveal positive cells corresponding to colonization by transplanted SSCs.
SSCs colonies have a typical grape-like morphology when cultured on mitomycin C-treated MEFs (Figs. 1A and 1B). For expansion of the SSCs cultured on feeder cells, feeders have to be prepared for every subculture. This approach has considerable drawbacks, as it involves several tedious and timeconsuming procedures, including culturing MEFs and their mitotic inactivation with mitomycin C. Therefore, a feeder-free culture system would allow maintaining and propagating SSCs in a labor- and cost-effective manner.
SSCs in the testis are located and maintained on the basal membrane of testicular tubules. This implies that the
To examine the expandability of SSCs cultured on Matrigel, we replated them at 5 × 105 cells per well of a 12 well plate every 5 days. The proliferation ability was maintained for more than 100 days at rates of approximately 2.5-fold per 5 days, which was similar to proliferation rates in the feeder culture system (Figs. 1E and 1F). This suggests that Matrigel can support unlimited
To characterize SSCs cultured in Matrigel-based feeder-free conditions, we performed RT-PCR and FACS analysis to examine SSC-specific gene expression at the mRNA (MEF and testis samples used as negative control and positive control, respectively) and surface protein levels, respectively. As shown in Fig. 2A, SSCs cultured under feeder-free conditions expressed the SSC marker genes
To investigate the epigenetic stability of SSCs cultured in the feeder-free system, we conducted bisulfite DNA sequencing analysis to determine the genomic imprinting pattern in these SSCs. DNA methylation patterns of the differentially methylated regions (DMRs) of the paternally imprinted gene
To determine the stem cell activity of cultured SSCs, we performed the spermatogonial transplantation assay. Because there is no clear functional
SSCs generally require the basement membrane to maintain their stem cell properties both
Several previous attempts have been made to culture SSCs under feeder-free conditions using laminin (Kanatsu-Shinohara et al., 2005; 2011), but in this case new SSC culture medium components were required. However, our feeder-free culture system uses the same SSC medium as the feeder culture systems, so no new components need to be prepared. Furthermore, we found an obvious difference in the SSC proliferation rate between laminin and Matrigel (Supplementary Fig. 1). Although it was not significantly different during the first 3 passages, SSC proliferation on laminin was dramatically reduced after 4 passages. This suggests that Matrigel is more suitable for maintaining proliferating SSC cultures
It should be noted that efficient propagation of SSCs on Matrigel requires more than 5 × 105 cells per well of a 12 well plate. Our feeder-free culture is cell number-dependent with the optimal cell density of approximately 1.3 × 105 cells/cm2. It is possible that the cell number-dependent growth of SSCs under feeder-free conditions may be related to the action of survivalenhancing autocrine factors, which is well described in ESCs (Mittal and Voldman, 2011).
We chose Matrigel as a matrix in our feeder-free culture for SSCs. Matrigel is the manufacturer’s trademark for ECM extracted from the Engelbreth-Holm-Swarm tumors (Vukicevic et al., 1992). In our initial experiments on feeder-free SSC culture, we used standard Matrigel that contains a mixture of ECM molecules and growth factors, such as EGF, IGF-1, TGF-β and PDGF. Later, we switched to growth factor?reduced (GFR) Matrigel for use in the feeder-free cultures. We found no difference between standard Matrigel and GFR-Matrigel in their ability to support SSC proliferation (Supplementary Fig. 3). This suggests that the growth factors in standard Matrigel may not influence SSC proliferation under feeder-free culture conditions, which was also confirmed by the results that adding IGF-1, TGF-β, and PDGF did not support proliferation of SSCs on gelatin-coated dishes. Thus, a combination of the major ECM components, such as laminin, collagen, entactin, and heparan sulfate proteoglycan, likely plays a role in creating proper basement membrane-like environment for SSCs in our feederfree culture.
Our expression analysis of genes for intracellular and cell surface SSCs markers revealed no differences between our feeder-free culture system and a feeder culture. DNA imprinting patterns of SSCs were also preserved in the feeder-free culture: the methylation status was androgenetic in both culture types. Furthermore, we found that SSCs transplanted to the male mouse testicular tubes were able to colonize the recipient testis. Therefore, the feeder-free culture system can maintain the cellular and molecular phenotypes as well as functionality of SSCs.
Our feeder-free culture system for expansion of SSCs is simple and efficient, because it uses the same SSC medium as the feeder cultures. Moreover, in comparison with the lamininbased culture, our Matrigel-based feeder-free culture system is superior as it allows long-term cell proliferation, whereas laminin cannot support SSC proliferation after 3 passages.
In this context, the Matrigel-based culture system provides time- and cost-effective unified approaches for feeder-free expansion of SSCs. Moreover, as it excludes variability caused by the presence of feeder cells, this system should be suitable for more definitive experiments to study the molecular mechanisms regulating SSCs, which is important in basic reproductive and stem cell biology, and also for therapeutic applications of germ cells in infertility treatment.
In this study, we developed a novel feeder-free culture system to support long-term maintenance of SSCs. We have demonstrated that our culture system preserves SSC properties properties at the molecular and cellular levels and their functionality. The advantage of our feeder-free culture system is that it provides a time- and cost-effective method to maintain SSCs, because it does not require feeders and uses the same medium as feeder cultures. Thus, our culture protocol will make culturing SSCs simple and reproducible, facilitate scale-up, and allow definitive experiments to evaluate individual factors affecting SSC stemness, which is important both in basic and applied reproductive biology.
Nari Hong and Yoonkey Nam
Mol. Cells 2022; 45(2): 76-83 https://doi.org/10.14348/molcells.2022.2023Seung-Won Lee, Guangming Wu, Na Young Choi, Hye Jeong Lee, Jin Seok Bang, Yukyeong Lee, Minseong Lee, Kisung Ko, Hans R. Sch?ler, and Kinarm Ko
Mol. Cells 2018; 41(7): 631-638 https://doi.org/10.14348/molcells.2018.2294Meeyoung Cho, Tae-Jun Cho, Jeong Mook Lim, Gene Lee, and Jaejin Cho
Mol. Cells 2013; 35(5): 456-461 https://doi.org/10.1007/s10059-013-0083-0