In vitro culture of CD9- and α6-integrin-expressing cells enriched by magnetic cell sorting from cryptorchid adult and pup testes in mice
Tasuku Mitani 1 , 2 *, Yoshiaki Ozaki 3 , Yusuke Tanaka 3 , Ayako Takeuchi 3 , Kazuhiro Saeki 1 , 3 , Hiromi Kato 1 , 2 , Kazuya Matsumoto 1 , 3 , Yoshihiko Hosoi 1 , 3 and Akira Iritani 1 , 2 , 3
Abstract
Recently, studies on cell surface markers of spermatogonia in combination with germ cell transplantation technique have made possible their functional analysis and the germline stem cells（GS cell）have established. GS cells are downstream of the stem cells such as ES cells and embryonic germ cells, namely EG cells, which are derived from PGCs. Therefore, GS cells are expected to be useful for the production of genetically-modified animals. In this study, we examined enrichment of GS cells expressing CD9 or α6-integrin from C57BL/6J cryptorchid adult and ICR pup（6-8 dpp）testes by magnetic cell sorting（MACS）and cultivation of those cells in vitro. Flow cytometric analyses demonstrated that MACS effectively enriched CD9-positive（CD9+）and α6-integrin-positive
（α6-integrin+）cells most effectively from pup testis. Therefore, CD9+ and α6-integrin+ cells from pup testes were used for the following cultivation. Cells proliferated in the first few days in suspension, subsequently attached to the culture plates and formed colonies after 3-5 days of culture. Those GS-like cells were passaged on the feeder layers and showed continuous proliferation for more than 2 months.
Immunocytochemical and FCM analyses demonstrated that those cells maintained the expression of CD9, α6-integrin and Oct4. These findings indicate that the CD9+ and α6-integrin+ cells collected from mouse pup testes have GS cell properties. Now, transplantation of GS-like cells into testis of recipient W mice which lack spermatogenesis is under way to evaluate their ability of spermatogenesis.
1 . Introduction
In mammalian germline, several kinds of stem cells have been established. Embryonic stem cells（ES cells）are originated from the inner cell mass cells in preimplantation embryos（1 , 2）and embryonic germ cells（EG cells）are also derived from PGCs（3）. Recently, germline stem cells（GS cell）have been established from spermatogonial stem cells（4）. The relationship of GS cells to ES and EG cells is physiological and（epi）genetic interests（5）. As spermatogonia undergo unique genetic and morphological changes to differentiate to functional gametes, GS cells are also anticipated to provide new strategies for producing genetically-modified animals in various species and for medical science.
Studies on cell surface markers have found several molecules specific to spermatogonia（6‑9）in combination with germ cell transplantation technique（10 , 11）which makes possible to analyze their function to generate spermatozoa. To date, CD9 and α6-integrin molecules are revealed to be specific to spermatogonia and using antibodies against these molecules they can be highly purified by cell sorting methods（6‑9）.
1. Institute of Advanced Technology, Kinki University and 2. Gene Control Corporation, Kainan, Wakayama 642-0017, Japan 3. Department of Genetic Engineering, Kinki University, Kinokawa, Wakayama 649-6493, Japan
In this study, with a view to further application to various animals, we examined enrichment and cultivation of mouse spermatogonial cells collected from cryptorchid adult testes（7 , 12 , 13）and pup testes（13）by magnetic cell sorting（MACS）method（14）. In the cryptorchid testis, which is hung inside the body at high temperature, differentiating spermatocytes are eliminated by apoptosis due to its body temperature（15 , 16）. In the pup testis until a week after birth, spermatogenesis has not yet started and such testis dominantly contains undifferentiated spermatogonia（17 , 18）. Enrichment of the cells expressing either CD9 or α6-integrin molecules were performed by MACS. In the first experiment, in order to examine the efficacy of enrichment of spermatogonia, MACS-fractionated cells were analyzed the expression of target molecules using a flow cytometry（FCM）. In the second experiment, we did in vitro culture of the cells.
2 . Materials and Methods
Preparation of testicular cells
Testis cells were collected from C57BL/6J（Clea-Japan）cryptorchid at 2-3 months after surgery
（Fig. 1 a）or non-treated adult testes and ICR（Clea-Japan）pup testes at 6-8 days postpartum（dpp）
according to previous reports（12 , 13）. In brief, testes were removed their capsule and digested by 0.1%
collagenase at 32℃ for 15 min, followed by 0.25% trypsin/0.53 mM EDTA at 37℃ for 15 min with gentle shaking. Dissociated cells were filtrated through glass-wool column followed by a cell strainer
（Falcon352340）（BD Bioscience, NJ）. They were then treated either with biotin-conjugated rat anti- mouse CD9 antibody（KMC8, BD Bioscience, NJ）or rat anti-human α6-integrin antibody（GoH3, BD Bioscience, NJ）, whose antigen is localized on the surface of spermatognia, followed by the streptavidin- microbeads（Miltenyi Biotec, Germany）or the anti-rat IgG-microbeads（Miltenyi Biotec, Germany）
treatment. The cell suspension was passed through a MACS-separation column according to manufacturer s instruction.
Indirect immunofluorescence
Cultured cells were fixed in 4% paraformaldehyde in PBS for 30 min at room temperature（RT）. Fixed samples were washed 3 times in PBS/0.1%BSA for every 10 min. Immediately before staining, the samples were incubated in blocking solution（5% skim milk in PBS）for 1 h at RT. After removing blocking buffer by washing in PBS/0.1%BSA 3 times for 10 min, they were incubated with biotin- conjugated rat anti-mouse CD9 antibody（KMC8, BD Bioscience, NJ）or rat anti-human α6-integrin antibody（GoH3, BD Bioscience, NJ）at a 1/200 dilution overnight at 4℃. The samples were again washed in PBS/0.1%BSA 3 times for 10 min. They were then incubated with the secondary antibodies, streptoavidin-FITC conjugate（BD Bioscience, NJ）or TRITC-conjugated rabbit anti-rat IgG antibody
（T-4280, SIGMA-ALDRICH, MO）at a 1/1000 dilution for 1 h at 4℃. After removing excess antibodies by washing in PBS/0.1%BSA 3 times for 10 min, immunostained samples were subsequently mounted with glycerol or mounting medium containing DAPI（H-1200, VECTASHIELD）. The specimens were analyzed using fluorescent microscopes.
Immunocytochemistry
Cultured cells were fixed in 4% paraformaldehyde in PBS for 30 min at RT. Samples were washed in PBS for 10 min, permeabilized by treatments in 0.1% Triton X-100 in PBS（0.1%PBT）3 times for 10 min, then quenched in 5% H2O2 in cold methanol for 30 min and washed 3 times in 0.1%PBT for 10 min. Samples were then incubated for 1 h in a blocking solution（5% skim milk in 0.1%PBT）at RT and washed 3 times in 0.1%PBT for 10 min. The samples were then incubated with goat anti-Oct4 polyclonal antibody（N-19, Santa Cruz Biotechnology, CA）at a 1/100 dilution overnight at 4℃. After washing 3 times in 0.1%PBT for 10 min, the samples were incubated with HRP-conjugated donkey anti- goat IgG polyclonal antibody（AP180P, CHEMICON, CA）overnight at 4℃, washed 3 times in 0.1%PBT for 10 min and stained using a HRP staining kit（Kirkegaard & Perry Laboratories）. The specimens were mounted with glycerol and analyzed using fluorescent microscopes.
Flow cytometric analyses
Flow cytometric analyses were performed on MACS-fractionated cells and cultured cells using a FACS- Calibur system（BD Biosciences, CA）. Briefly, MACS-fractionated cells and cultured cells were suspended in 0.1 ml of PBS/1%FBS. To identify CD9-positive cells, the cells were incubated with biotin- conjugated rat anti-mouse CD9 antibody（KMC8, BD Bioscience, NJ）at a 1/200 dilution for 1 h at RT, and subsequently washed twice in PBS/1%FBS. The cells were then incubated with streptoavidin-FITC conjugate（554060, BD Bioscience, NJ）as the secondary antibody at a 1/1000 dilution for 1 h on ice.
To identify α6-integrin-positive cells, the cells were treated with rat anti-human α6-integrin antibody
（GoH3, BD Bioscience, NJ）at a 1/200 dilution followed by TRITC-conjugated rabbit anti-rat IgG antibody（T-4280, SIGMA-ALDRICH, MO）at a 1/500 dilution in the similar way. To remove excess FITC- or TRITC-conjugated antibody, immunostained cells were washed twice in PBS/1%FBS and suspended with 1 ml of PBS/1%FBS. Cell suspension was subsequently analyzed using FACS Calibur.
Cell culture
MACS-fractionated cells were cultured on 0.1% gelatin-coated MultiDish（176740, Nunc）with 1-2 x 105 cells/well in a culture medium at 37℃ under 5%CO2 in air according to the previous report（4）. Well-grown cells were dissociated by 0.25% trypsin/0.53 mM EDTA treatment and plated on mitomycin C-inactivated primary mouse embryonic fibroblast cells（termed feeder cell layers）. Culture medium was StemPro34-SFM（Invitrogen, CA）supplemented with StemPro supplement（Invitrogen, CA）, 1%
FBS, BSA（5 mg/ml）, MEM-vitamin solution（Invitrogen, CA）, MEM-non essential amino acids（Invitrogen, CA）, mouse leukemia inhibitory factor（mLIF）（ESGRO, 103U/ml）（CHEMICON, CA）, rat glial cell- derived neurotrophic factor（GDNF）（10 ng/ml）（PeproTech, UK）, bovine basic fibroblast growth factor（bFGF）（10 ng/ml）（PROGEN Biotechnik, Denmark）, mouse epidermal growth factor（EGF）
（20 ng/ml）（PeproTech, UK）, ascorbic acid（10‑4M）, d-biotin（10μg/ml）, putrescine（60μM）, β-estradiol（30 ng/ml）, progesterone（60 ng/ml）, D-glucose（6 mg/ml）, pyruvic acid（30μg/ml）, DL-lactic acid（1μg/ml）and L-glutamine（2 mM）.
3 . Results
Enrichment of CD9- or α6-integrin-expressing cells from non-treated, cryptorchid and pup testes by MACS.
To determine conditions for preparation of spermatogonia from testes using cell surface markers, MACS-fractionated cells were analyzed by FCM on enrichment of CD9- or α6-integrin-positive cells from non-treated adult, cryptorchid adult and pup testes. Immunofluorescent staining visualized enrichment of the cells expressing both CD9 and α6-integrin in CD9-MACS fraction（Fig.1b）. Flow cytometric analysis was then performed to measure the efficacy of MACS for enrichment of CD9 and α6-integrin expressing cells. In the case of non-treated adult testis, CD9 expressing cells were apparently enriched in CD9-selected MACS fraction（Fig. 1c, upper）, but CD9-negative cells still dominantly retained there. On the other hand, in the case of cryptorchid testis, it was shown drastic decrease of CD9 negative cells in MACS fractionated population（Fig.1c, lower）. Pup testes were also examined in the similar way. As shown in Fig. 1d, both in the case of CD9 and α6-integrin selection by MACS, the cells expressing those molecules were quite effectively separated from negative cells for CD9
（Fig.1d, middle）or α6-integrin（Fig.1d, right）. These results indicate that spermatoginia were enriched most effectively from pup testes. Therefore, pup testes were used to collect sprematogonia for in vitro culture.
Morphological and immunocytochemical analyses of α6-integrin selected cells cultured in vitro.
MACS fractionated cells were then cultured on gelatin-coated plates at 37℃. The cells initially floated for a few days and then gradually settled down on the culture plate. They loosely attached on flat somatic cells which were mixed in MACS selected fraction and began colonization after about 5 days of culture. After about 2 weeks, well-grown cells were allowed to be passaged onto feeder cell layers
（Fig. 2a-c）. Thereafter, the cells were passaged onto fresh feeder cell layers every 7 to 10 days
（Fig. 2d , j）. In this culture, expression of CD9, α6-integrin and Oct4 was examined by FCM and immunocytochemistry. As shown in Fig.2e-i, the expression of CD9 and α6-integrin maintained in the primary culture cells on 14 days of culture and Oct4 expression was also observed in those colonized cells. These colonies were then dissociated and passaged onto another plates with feeder cell layers.
The cells colonized again and showed CD9, α6-integrin and Oct4 expressions on 21 days of culture
（Fig.2k-o）and those molecules were still expressed after one month of culture（not shown）.
Flow cytometric analysis of CD9- and α6-integrin-selected cells cultured in vitro.
To evaluate the expression profile of CD9 and α6-integrin in long term culture, the cells derived from α6-integrin-selected cells were examined by FCM. Expression of both CD9 and α6-integrin on cultured cells was maintained for at least 20 days（Fig. 3a）. Further expression of CD9 in α6-integrin-selected cells was confirmed for at least about 2 months in the cells derived from CD9-selected cells（Fig.3b）.
These results indicated that cultured cells maintained undifferentiated spermatogonial characteristics.
Fig. 1． Flow cytometric analysis of MACS-treated fractions on enrichment and cellular characteristics.
（ a ）Appearance of cryptorchid testis 2 months after surgery. The size of cryptorchid testis（left）
appears small compared to normal testis（right）.（ b ）Fluorescent images of cells fractionated by MACS using anti-CD9 antibody followed by cytospin preparation.（ c , d ） Enrichment of CD9+ and α6-integrin+ cells from non-treated, cryptorchid and pup testes by MACS. Comparison of purification of CD9+ cells between non-treated and cryptorchid adult testes（ c ）. Although drastic decrease of CD9-negative cells in MACS fractionated population was shown in cryptorchid testis, they still retained in MACS fraction in half. On the other hand, both in the case of CD9（ d , middle）and α6-integrin（ d , right）selections by MACS, the cells expressing those molecules were quite effectively separated from negative cells in pup testis.
Fig. 2． In vitro culture of α6-integrin+ cells collected from pup testes.（ a - d and j ）Morphology of α6-integrin-selected cells in in vitro culture. Plated cells（ a , at 1 day cultured in vitro
（1 DIV））progressed cell division slowly（ b , at 3DIV）and formed colonies（ c , at 14 DIV）.
D i s p e r s e d c e l l s （ d , a t 1 5 D I V ） c o l o n i z e d a g a i n （ j , a t 2 0 D I V ）.（ e - i a n d k - o ） Immunocytochemical analysis on expressions of CD9, α6-integrin and Oct4 in cultured cells at 14 DIV（ e - i ）and 21 DIV（ k - o ）. Colonies at 14 DIV consisted of the cells with euchromatin strongly stained with DAPI（ e , h ）expressed CD9（ f ）and α6-integrin（ i ）.
Colonies like bunch at 21 DIV（ k , n ）expressed CD9（1）and α6-integrin（ o ）. Expression of Oct4 was detected in nuclei of the colonized cells at 14 DIV（ g ）and 21 DIV（m）.（Scale bar; 25μm）
4 . Discussion
Our results show that MACS system could effectively enrich the cells expressing either CD9 or α6-integrin molecule and that MACS-fractionated cells could proliferate with undifferentiated sprematogonial characteristics for long-term on the feeder cell layers, as demonstrated in other studies
（4 , 5 , 9 , 19 , 20）. Regarding to preparation of spermatogonia, pup testes showed the most effective enrichment rather than cryptorchid testes. This might be because spermatogenesis in pup testis has not yet started. In male germline in mice, primordial germ cells（PGCs）first appear at embryonic day 7
（7dpc）in the extraembryonic region named allantois（21）. Then they migrate into the genital ridge and form gonads with stromal cells by 11.5 dpc. Male PGCs proliferate mitotically in the gonads and subsequently enter G0/G1 arrest until birth. Just after birth（0.5 dpp）, arresting germ cells
（gonocytes）resume mitosis and by several days after birth they locate on the basement membrane in seminiferous tubule to develop into spermatogonial stem cells（SSCs）. In the previous reports, GS cells were established from neonatal testis（4 , 5）, but in these cases, there was still a possibility that they were Fig. 3． Flow cytometric analysis of expression of CD9 and α6-integrin on MACS-selected cells
cultured in vitro.（ a ）Cultured cells for 20 DIV in passage-1 maintained expression both of CD9（left）and α6-integrin（right）.（ b ）Cultured cells at 27 DIV（left）and 58 DIV（right）
demonstrated the expression of CD9. Controls ; The cells treated only with FITC-conjugated 2nd antibody.
derived from PGCs which remained in neonatal testis or gonocytes. Kubota et al.（9）showed that GS cells were derived from SSCs in pup testis at 4.5-7.5 dpp enriched by MACS for Thy-1 which is expressed on SSC. Although we have not yet examined the potency of the cultured cells to generate sperm by transplantation into recipient testis, our results indicate that enrichment of spermatogonia by MACS for CD9 or α6-integrin successfully derived GS cells from pup testis. We also demonstrated that cryptorchid adult testis effectively enriched sprematognia in vivo but it seemed to be still inadequate for purifying spermatogonia via MACS（Fig. 1b）. On the other hand, the previous report by Ogawa et al.（18）demonstrated that GS cells could be established from spermatogonia collected from adult testis by enrichment by serial recovery of floating cells left overnight on gelatin-coated plates. It was estimated that the ratio of SSCs in adult testis is quite low, 0.01% of germ cells in the testis（22）. Nevertheless, adult testis contains spermatogonia quite more than pup and neonatal one. Therefore, adult testis is thought to be preferable source for collecting SSCs. The combination of cryptorchid testis, MACS and gelatin-coat treatment might develop more effective enrichment of spermatogonia from adult testis.
When MACS-fractionated cells were plated on gelatin-coated culture plates, somatic cells which had remained in the MACS column attached and grew faster than spermatogonia and might act on spermatogonia as supporting cells. On those somatic cells, spermatogonia attached loosely and started cell division to form colonies. It was noted that appearance of the colonies was quite different from that of embryonic germ（EG）cells and rather the cells showed round shape and clear outline as shown in Fig. 2. This observation might suggest that they were not derived from PGCs. Those colonies were allowed to be dispersed by trypsinization. The dissociated single cells started cell division on the feeder cell layers and proliferated in the similar way for long-term and they maintained expression of CD9 and α6-integrin beyond several passages. Now, transplantation of those cells into testis of WBB6F1W/Wv mice, which lack spermatogenesis due to its defect of c-kit gene, is under way and the potency of the cultured cells in the present study would be evaluated in the near future. Recently, it has been reported that, although GS cells itself was restricted its ability only to differentiate to sperm, they happened to change their morphology like ES cells and subsequently acquired multipotency to generate chimeric mice by injecting into blastocysts. This ES-like cells were named multipotent GS（mGS）cells（5）. This transformed mGS cells have been always generated at least about 2 months after culture. We have already cultured GS-like cells for about 2 months, but such ES-like colonies have not yet appeared.
GS cells have quite valuable features both for basic and applied science and technology. In the study of gametegenesis, GS cells provide a unique in vitro model system to figure out its molecular mechanisms, which would be helpful for clinical application. For example, gene silencing technology by RNA interference（RNAi）in ES cells provide a novel strategy to produce knockdown mice and knockdown mice of superoxide dismutase 1（SOD1）, which mutation induces autosomal dominant disease, familial amyotrophic lateral sclerosis（ALS）, demonstrated drastic prevention of the development of familial ALS（23）. As lots of genes expressed during spermatogenesis remain to be solved their function, gene silencing by RNAi in GS cells would provide powerful tool to analyze the function of certain target genes. Other genetic manipulation techniques such as homologous recombination and viral vectors（24 , 25）would be also available for GS cell manipulation. Such genetic manipulations in GS cells would be applied for producing transgenic animals in combination with
transplantation into testis. Theoretically, after genetic manipulation with GS cells, genetically-modified sperm would be generated in half, which would result in 50% of offspring to be transgenic when W/Wv mice were used as recipients. Indeed, using this strategy, Shinohara et al.（26）demonstrated the production of transgenic mice by natural mating with efficacy of about 50%. Therefore, it is expected that optimization of culture condition of spermatogonia would give rise to GS cells as a novel material for genetic manipulation in various species including cattle（27 , 28）. However, so far, it still remains impossible to induce spermatogenesis in other species except rat and hamster completely in mouse testis after transplantation although they could survive in recipient testis for more than several weeks
（29‑32）. This might be because of incompatibility of cytokines and some other molecules between germ cells and their environment rather than immunological reactions（33）. To overcome this difficulty, induction of spermatogenesis in vitro at least until producing haploid germ cells could be useful. Finally, again, establishment of GS cells and induction of their spermatogenesis in other species represents an essential tool that will provide answers to many questions and enhance progress in the life sciences.
Furthermore, this fascinating technology is surely to develop new horizon in clinical and industrial applications in future.
5 . Acknowledgements
We appreciate technical suggestions from Dr. T Ogawa at Yokohama City University. This work was supported by the Wakayama Prefecture Collaboration of Regional Entities for the Advanced of Technological Excellence, JST, by a Grant-in-Aid for the 21st Century Center of Excellence Program of the MEXT, Japan, and by a Grant-in-Aid for Scientific Research（15580251）from the Japan Society for the Promotion of Science.
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和 文 抄 録
マウス実験的停留睾丸および幼若精巣から磁気細胞分離法により濃縮した CD9 ならびにα6 インテグリン発現細胞の体外培養
三 谷 匡、尾 崎 嘉 昭、田 中 裕 介、
竹 内 礼 子、佐 伯 和 弘、加 藤 博 己、
松 本 和 也、細 井 美 彦、入 谷 明
近年、マウスにおいて精子幹細胞（GS 細胞）の樹立が報告されている。GS 細胞は ES 細胞や始原生殖細 胞から派生する EG 細胞のさらに下流に位置する幹細胞であり、遺伝子改変動物の作製に応用できると期 待される。本研究では、分化精子細胞の除去処理（実験的停留睾丸）成体マウス精巣および精子形成がま
だ始まっていないマウス新生仔精巣より、CD9あるいはα6 インテグリンの発現を指標に磁気細胞分離シ
ステム（MACS）により精子幹細胞を濃縮し培養を行った。フローサイトメトリー（FCM）を用いて
MACS 分画の回収率、濃縮率等を解析した結果、MACS によりマウス精巣からの CD9、α6 インテグリン
発現細胞の濃縮効果が示された。また、実験的停留睾丸では CD9陰性細胞集団の混入が多く、幼若マウ
ス精巣では少ないことが示された。そこで幼若マウス精巣より分離濃縮した CD9あるいはα6 インテグ
リン発現細胞の体外培養を行った結果、培養開始後数日間は浮遊状態であったが、3 〜 5 日でコロニーの 形成が見られた。増殖した細胞を継代培養した結果、播種した細胞は増殖を続け、さらに免疫組織化学お よび FCM 解析より、CD 9、α6 インテグリンならびに未分化細胞の特徴的な転写因子である Oct 4 の発 現が継続して観察された。現在、培養条件の適正化とともに、これらの細胞の精子形成能力について精巣 移植法を用いて機能評価を進めている。