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STEM CELLS IN THE NEWS |
1 Executive Director, Cell Therapy R & D Head, Hematopoiesis Department Holland Laboratory, American Red Cross hawleyr{at}usa.redcross.org
2 Research Communications Manager Holland Laboratory, American Red Cross sobieski{at}usa.redcross.org
"So come up to the lab and see what’s on the slab. I see you shiver in antici...pation."
Richard O’Brien, "The Rocky Horror Picture Show"
A spate of recent articles describe new assisted reproductive technologies that are being developed which may find immediate application in the preservation of endangered wildlife and the management of commercial livestock. Some of these technologies could conceivably be used to provide improved treatment modalities to overcome fertility problems in humans, but their potential implementation to establish human pregnancies would raise broad social, ethical, legal, and philosophical issues rivaling those currently surrounding embryonic stem cell research and human cloning [1–3].
SPERMATOGONIAL CELL LINE
It is difficult to maintain viable spermatogonia for long periods of time in culture, stymieing efforts of in vitro sperm production. Feng et al. [4] reported in the July 19 issue of Science that in the presence of stem cell factor (SCF, c-kit ligand) telomerase-immortalized mouse type A spermatogonia—male germline stem cells—give rise to spermatocytes and spermatids in vitro. Although high telomerase activity characterizes type A spermatogonia, that activity is lost progressively during differentiation. Therefore, the investigators transduced undifferentiated type A murine spermatogonia with a retroviral vector containing the cDNA for murine TERT (telomerase reverse transcriptase), the telomerase catalytic component, together with the bacterial neo gene. After 2 months of culture under G418 selection, surviving cells continued to proliferate and became immortalized. Cells of the resulting six clonal lines (designated S1 to S6) exhibited a morphology resembling primary type A spermatogonia and were observed to express c-kit—the receptor for SCF and a biochemical marker for spermatogonia—and other proteins characteristic of primordial germ cells.
The researchers assessed the developmental potential of S4 cells by evaluating SCF-mediated meiosis. SCF stimulated phosphorylation of c-kit and synaptonemal complex formation as revealed by the expression of synaptonemal complex protein 3, a protein associated with the chromosomal reorganization caused by meiosis. The investigators then transfected S4 cells with the green fluorescent protein (GFP) gene under the control of the acrosin promoter—activated in the cytoplasm of stage IV pachytene spermatocytes—and treated them with SCF. The S4 cells appeared to differentiate into spermatocytes and round spermatids, with GFP+ proacrosomal granules and acrosomes observed, respectively. The expected pattern of chromosome number was also observed during SCF-induced differentiation: one week after SCF treatment, the S4 cells showed a tetraploid karyotype whereas 2 weeks later, slightly more than half of the cells were haploid. Thus, studies of TERT-immortalized type A spermatogonial stem cells may provide insights into the molecular mechanisms of spermatogenesis, facilitating systematic analysis of the requirements necessary to establish long-term cultures of primary spermatogonia.
SPERM FACTORIES
In the August 15 issue of Nature, Honaramooz et al. [5] reported the production of mature sperm in testis tissue grafted from newborn mice, pigs, or goats into immunodeficient mouse hosts. Small fragments (0.5-1 mm3) of testis tissue from neonatal mice, pigs, or goats were grafted under the back skin of castrated recipient mice. The researchers euthanized recipients at 2-to-4-week intervals and measured androgen production as reflected in blood hormone levels, assessed the growth and development of the transplanted tissue, and observed the progression of spermatogenesis. They found that over 60% of the grafts from all three donor species survived, showing up to 100-fold growth, regulating steroidal production and eventually producing mature sperm cells that were capable of fertilization. In addition, the grafts were observed to fully supplement androgens to the castrated recipients. Recipient mice showed no sign of neoplasia or other disorder as long as a year after the transplant.
When neonatal mouse tissue was transplanted, spermatogenic progression precisely mimicked that in intact mouse testis although in many seminiferous tubules, there was a dilation of the lumen and a disorganized epithelium which the researchers attributed to lack of fluid flow. Xenografted pig testis tissue showed complete spermatogenesis that commenced 2 weeks earlier than in pig testes, but followed the developmental pattern expected for pigs. Transplantation of goat testis tissue also resulted in the production of mature, viable sperm. Sperm recovered from grafted tissue from all three species were capable of fertilization after intracytoplasmic injection into mouse oocytes. Homologous mouse embryos produced using this approach that were transplanted to pseudopregnant mice showed normal fetal development. The team also investigated the effects on the transplants of 2 days of cooling or longer periods of cryopreservation of the collected neonatal tissues. Such storage did not impede the ability of the tissues to fully support spermatogenesis and steroidogenesis when grafted into mice.
Previous efforts to transplant spermatogonial stem cells from phylogenetically distant species into mouse testes failed to establish spermatogenesis beyond the stage of spermatogonial proliferation. The investigators suggest that the testis tissue grafting procedure described could be used to help maintain endangered species or valuable livestock by allowing sperm production from immature males, as a model for toxicology testing on testicular tissue and sperm, as a tool for the further study of germ cell development, and for the production of sperm from animals with genetic conditions that prevent survival to sexual maturity. They also raised the possibility that the xenograft approach may be a means to provide sperm for assisted in vitro fertilization in humans as an alternative to restoring fertility in an individual following cancer therapy.
OOGENESIS AND OVARY TRANSPLANTS
In the August 1 issue of Nature, Obata et al. [6] described the first successful in vitro culturing of immature oocytes—female germline stem cells—into mature oocytes that could be fertilized to give rise to live newborns. The investigators cultured ovaries from mouse fetuses at 12.5 days post coitum, but observed that the oocytes that developed during the 28-day culture period were unable to resume meiosis. This process was completed by transferring the nuclei into enucleated, fully grown oocytes from adult mice, indicating that nuclear reprogramming is essential for the in vitro production of totipotent zygotes.
In a study published earlier this year (in the January 24 issue of Nature), Wang et al. [7] reported a qualified success in preserving reproductive potential following transplantation of cryopreserved ovarian tissue in rats. The team dissected the right ovary and reproductive tract, including the upper segment of the uterus and transplanted them as a unit into syngeneic recipients. In the control operation, the tissue was held in solution for an hour before transplant; the experimental organs were perfused with M2 medium and increasing concentrations of dimethylsulphoxide (DMSO) to a concentration of 1.5 M then cooled to -7°C and stored in liquid nitrogen overnight. The next day, frozen organs were rapidly thawed and perfused to remove DMSO. The animals were monitored for ovarian function for 10 weeks and some were paired with stud males. The animals were then euthanized, their blood collected for measurement of follicle-stimulating hormone (FSH) and estradiol, and the transplanted organs prepared for histology. Animals that received fresh transplants reinitiated estrus and showed normal numbers of ovarian follicles, normal uterine weight, and FSH levels close to those of untreated controls. Four of seven animals with cryopreserved grafts had ovarian follicles and corpora lutea, and showed higher FSH serum levels and lower estradiol levels compared to the group that received fresh organs. One of the animals that received cryopreserved organs was found to be pregnant with two normal fetuses. The authors stated that although ovarian function is diminished by freezing, the results overall are encouraging and suggest that frozen banking of reproductive organs may someday be helpful in breeding endangered species or may restore fertility in females who have undergone sterility-inducing therapies [8].
A complementary report by Snow et al. published in the September 27 issue of Science [9] described an alternative method to preserve ovarian function involving xenografting. Ovaries from 3-week-old FVB mice were halved and grafted under the kidney capsule of adult male or ovariectomized female immunodeficient nude rat recipients. Twenty-one days after grafting, germinal vesicle stage oocytes and expanding cumulus oocyte complexes were collected from the xenografts, matured in vitro for 18 hours, and then fertilized with sperm from FVB male mice. Significantly more ovarian xenografts were recovered from female than male recipients: 83.3% treated with pregnant mare serum gonadotrophin and 78.9% without treatment versus 9.1% in treated males. Of these 57.9%, 40.0%, and 20.0%, respectively, formed two-cell embryos, with 9.4%, 6.1%, and 0% of these embryos resulting in live births following transfer to pseudopregnant females. The authors suggested that it may be possible with this technique to salvage ovarian tissue from recently deceased animals to aid in the propagation of rare species, but raise a cautionary note about applying this strategy to humans.
TRANSGENESIS BY TESTIS CELL TRANSPLANTATION
Brinster and colleagues recently published several articles describing genetic modification of spermatogonial stem cells [10–12]. In a proof-of-principle study that appeared last year in the Proceedings of the National Academy of Sciences, the group demonstrated that these genetically modified testis cells were capable of generating transgenic animals [12]. The investigators prepared testis cells from donor pups (C57BL/6 x 129SvCp [B6/129] F2 hybrid mice or C57BL/6 [B6] mice) and donor B6 mouse testes of 3- to 4-month-old males made cryptorchid at 6 to 8 weeks of age. They cultured the cells for 3 days, exposing the cells repeatedly to medium containing a recombinant retrovirus carrying the Escherichia coli lacZ gene under the control of the Pgk-1 promoter. The researchers then injected 1.2 x 105 to 4 x 105 cultured cells into each testis of recipient W-mutant (c-kit-defective) pups or B6/129 F1 hybrid pups. Recipient males were mated to wild-type females and the offspring were tested for expression of the lacZ gene by X-gal staining of ear or tail samples. Notably, transplantation of transduced spermatogonial stem cells restored fertility to infertile W-mutant males. Mating and progeny analysis was continued for 250 to 390 days after cell transplantation. Subsequently, recipient males were euthanized, and their testes were removed and sections were stained with X-gal and examined histologically to demonstrate spermatogenesis arising from the modified stem cells. In addition, chromosomal DNA of some of the progeny was analyzed to determine the integration site of the viral transgene.
The researchers found that their procedure for retroviral gene delivery did yield effective transduction, and they observed that stem cells from donor pup testes were more efficiently transduced (20% of cultured cells survived and were transduced) than male germline stem cells from cryptorchid adult donors (only 2.3 % of cultured cells survived and were transduced). They also observed that wild-type recipients gave rise to wild-type progeny only and did not show any X-gal-stained colonies on analysis, indicating that endogenous spermatogenesis prevents establishment of sperm-producing colonies from transduced cells. The low percentage (4.5%) of progeny that carried the transgene implied that most transduced stem cells contained a single copy of the virus. This was confirmed by Southern blot analysis, and stable transmission of functional transgenes to progeny was demonstrated for two lines. Interestingly, the different immunological backgrounds of the donor and recipient mice did not appear to impede spermatogenesis, suggesting that the microenvironment of the testis may allow allogeneic spermatogonial stem cell transplantation without need for immunosuppression.
The authors discussed the potential advantages of utilizing male germline stem cell transduction as a method of generating transgenic animals. Specifically, they suggested that because spermatogenesis is an ongoing process in the mature male and technology for cryopreserving sperm is well established, it would be possible to derive a large number of transgenic offspring from a single recipient of transduced spermatogonial stem cells.
RESTORATION OF SPERMATOGENESIS BY SERTOLI CELL GENE TRANSFER
In a recent issue of Proceedings of the National Academy of Sciences, Ikawa et al. [13] presented an alternative method for restoring spermatogenesis in infertile male mice using lentiviral vectors to transduce somatic Sertoli cells. The researchers first tested the transduction efficiency of a variety of recombinant viral vector platforms—adenovirus, adeno-associated virus type 2, murine leukemia virus, human immunodeficiency virus (HIV)—carrying the lacZ gene under control of the cytomegalovirus (CMV) immediate early region promoter by injecting the vectors into the seminiferous tubules of wild-type mice. They found that of all vectors tested, HIV-based lentiviral vectors yielded good transduction and successful expression of the reporter gene as evidenced by X-gal staining, did not have toxic effects on testicular cells, and did not interfere with subsequent breeding of recipients. The researchers used polymerase chain reaction to analyze the genomic DNA of 390 progeny of recipient mice and did not detect germline transmission of the lacZ gene.
The investigators then injected a lentiviral vector carrying the SCF gene (lack of which causes failure of spermatozoa to mature in male Sl/Sld mutant mice) under the control of the CMV promoter into seminiferous tubules of Sl/Sld mutant mice. The recipients were euthanized at various times after treatment, and it was found that spermatogenesis had been restored in all of the nine recipients examined. The researchers used Western blot analysis to detect SCF in the treated testes. Although spermatogenesis was restored by transduction of the mutant mice, the recipients were unable to produce pups by normal mating, and the investigators could not obtain enough sperm for standard in vitro fertilization. The investigators were, however, able to obtain offspring of the recipients by using intracytoplasmic injection of spermatogenic cells into wild-type ova. Of 90 oocytes injected, 59 developed normally in vitro, and 13 progeny were obtained after transfer of the developing embryos to pseudopregnant females. The offspring carried either the Sl or Sld locus in tandem with a wild-type allele, but none were transgenic for the lentiviral vector or the SCF gene, reinforcing the low probability of germline transmission using this system.
The investigators hypothesized that germline transmission is precluded because the Sertoli cells cover spermatogenic cells and remove the lentiviral particles in the seminiferous tubule lumen by endocytosis, preventing the vectors from reaching spermatogenic cells in vivo. The tight cell junctions between Sertoli cells may also help in preventing lentiviral transduction of spermatogonia. The authors concluded by stating that no animal studies have shown germline transmission after in vivo viral vector treatment and proposed that a lentiviral vector-mediated transduction system that allows gene transfer to Sertoli cells may provide a basis for gene therapy of some forms of male infertility. They did note, however, that because it is difficult to totally rule out the possibility of germline transmission, further large scale and detailed studies may be required should clinical application be contemplated. This would appear to be warranted in view of recent evidence that spermatogonial stem cells are not intrinsically resistant to transduction by lentiviral vectors [11].
THE ORIGIN OF TEENAGE MUTANT NINJA TURTLES®
As for the origin of Michaelangelo, Donatello, Raphael, and Leonardo (http://www.ninjaturtles.com), it apparently had nothing at all to do with germ cells—neonatal, genetically modified or otherwise—but rather involved "transdifferentiation" of somatic cells!
DISCLAIMER
Any views and opinions expressed herein are those of the authors. They do not necessarily reflect the policies or position of the American Red Cross.
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