[Stem Cell Studies 2011; 1:e15] [page 91] In vitro and in vivo lineage conversion of bone marrow stem cells into germ cells in experimental Azoospermia in rat Mohammed T. Abdel Aziz, 1 Taymour Mostafa, 2 Hazem Atta, 1 Soheir Asaad, 3 Hanan Fouad, 1 Gamal Mohsen, 1 Laila Rashed, 1 Dina Sabry, 1 Mohammed Abbas 1 1 Unit of Biochemistry and Molecular Biology Medical Biochemistry; 2 Andrology & Sexology; 3 Histology Departments, Faculty of Medicine, Cairo University, Cairo, Egypt Abstract The present study is conducted to assess in vitro transdifferentiation of bone marrow derived mesenchymal stem cells (MSCs) into germ cells and in vivo transdifferentiation into spermatids and spermatocytes in the seminif- erous tubules of azoospermic rats. Forty eight male rats were included in the study and were divided into: group 1; control rats, group 2; experimental azoospermic rats, group 3; azoospermic rats received undifferentiated MSCs. Group 4; azoospermic rats received transdifferentiated MSCs into germ cells. Quantitative real time PCR was conducted to assess gene expression of a primordial germ cell marker (VASA), stem cell specific markers (Oct4, SSEA-1 and SSEA-3), specific molecular markers of germ cells (c-Kit, Daz1); premeiot- ic marker (Daz1) and post-meiotic markers (c- Kit, Stra 8). Histopathological examination of rat testicular tissue was also conducted. Results revealed that 12 weeks after transplan- tation, fluorescent labeled MSCs were detected in the seminiferous tubules of the testes of group 3 and group 4 rats. In group 3 and 4, stem cell specific markers; Oct4, SSEA-1 and SSEA-3 were detected. In group 4, genes of pri- mordial germ cell marker; VASA, premeiotic marker; Daz1and post-meiotic markers; c-Kit, Stra 8 were expressed. Daz1 was also expressed in group 3 rats to a significantly lower extent in comparison to group 4 rats. Spermatocytes and spermatids were detected in testicular tissue of group 4 rats. In conclu- sion, MSCs have potentials for in vitro transd- ifferentiation into germ cells and in vivo trans- differentiation into spermatids and spermato- cytes. Introduction Spermatogenesis is generated by male ger- minal stem cells as evidenced by regeneration of spermatogic cells after transplantation of total testicular germ cells in the depleted testis. 1,2 Several studies have assessed the potential of reproducing germ cell differentia- tion, or gametogenesis, in vitro. 3 Under appro- priate conditions, bone marrow stem cells (BMCs), embryonic stem cells (ESCs) could be induced to transdifferentiate toward the germ cell lineage. 4-6 Several breakthroughs discovery concerning the in vitro derivation of male and female germ cells from stem cells have challenged the field of reproductive biology. 7-10 Mouse bone marrow mesenchymal stem cells (MSCs), grown in vitro in the presence of retinoic acid, were found to express germ cell markers. 11,12 A similar transdifferentiation process was described for human bone marrow cells. 13,14 Nayernia et al. 15 stated that BMS cell- derived germ cells expressed the known molec- ular markers of primordial germ cells; fragilis, stella, Rnf17, Mvh and Oct4; and molecular markers of spermatogonial stem cells and spermatogonia; Rbm, c-Kit, Tex18, Stra8, Piwil2, Dazl, Hsp90alpha, beta1- and alpha6- integrins. Another study has demonstrated that adult bone marrow stem cells can differen- tiate into Leydig cells in rat testes. 9 Lue et al. 12 provided evidence showing that adult bone marrow cells injected into seminiferous tubules or interstitial spaces of busulfan treat- ed mice are able to differentiate into germ cells as evidenced by the detection of VASA marker and they are also able to differentiate into Sertoli and Leydig cells as evidenced by the detection of follicular stimulating hormone receptors and cytochrome P450 (P450scc) side chain cleavage enzyme within the seminifer- ous tubules. The present study was conducted to assess transdifferentiation potentials of MSCs into germ cells in vitro and transdifferentiation potentials of MSCs into spermatids and sper- matocytes in the seminiferous tubules of azoospermic rats. Materials and Methods Animal studies The in vivo study was conducted on forty eight Cur1:HEL1 male albino rats, of an aver- age weight 180-200 gm, age 60 days. Rats were inbred strain in the experimental animal unit, Faculty of Medicine, Cairo University. Animals were maintained in an air-conditioned animal house with specific pathogen-free conditions, and were subjected to a 12:12-h daylight/dark- ness and allowed unlimited access to chow and water. They were maintained according to the standard guidelines of Institutional Animal Care and Use Committee and after Institutional Review Board approval. Rat were equally divided into the following groups: group1 involved control rats received phosphate buffer saline, group 2 included rats with experimental azoospermia induced by administration of a single intraperitoneal dose of busulfan (GlaxoSmithKline Inc., Mississauga, Ontario, Canada) (35 mg/kg), group 3 included azoospermic rats that under- went transplantation of 1×10 8 undifferentiated MSCs into the testis 2 weeks after induction of azoospermia. Group 4 involved azoospermic rats that underwent transplantation of 1×10 8 differentiated MSCs into germ cells 2 weeks after induction of azoospermia. The injection procedure was conducted as previously decribed. 16 All recipient rats were kept under anesthesia by intraperitoneal administration of Xylazine (20 mg/kg), Ketamine (5 mg/kg), and Athropine (0.04 mg/kg). Microinjection needles 100 μL were used for MSCs transplan- tation. The testes were surgically exteriorized through a midline abdominal incision, and MSCs suspensions (1×10 8 ) were injected using microinjection needles. Small incision was made 5 mm from the efferent bundles’ junction with the testis. The tip of the microin- jection needle was inserted into the bundles and then gently pushed toward the rete testis to enter the seminiferous tubules as described by Ogawa et al. 16 Twelve weeks after MSCs transplantation, animals were euthanized and testicular tis- sues were collected from all animals and the following parameters were assessed: Testicular histopathological examination, detection of the labeled bone marrow derived stem cells by using PKH26 Red Fluorescent Cell Linker Kit (Sigma Aldrich, St. Louis, MO, Stem Cell Studies 2011; volume 1:e15 Correspondence: Hanan H. Fouad, Medical Biochemistry Department, Faculty of Medicine, Cairo University, Cairo, Egypt, POB 11562. Tel. +20.101.418.750 - Fax: +20.223.632.297. E-mail: hananfouadbostamy24@gmail.com Key words: azoospermia, germs cells, MSCs, spermatids, spermatocytes. Received for publication: 3 September 2011. Accepted for publication: 11 October 2011. This work is licensed under a Creative Commons Attribution NonCommercial 3.0 License (CC BY- NC 3.0). ©Copyright M.T. Abdel Aziz et al., 2011 Licensee PAGEPress, Italy Stem Cell Studies 2011; 1:e15 doi:10.4081/scs.2011.e15 Non-commercial use only