Generation of lung epithelial-like tissue from human embryonic stem cells.

BACKGROUND: Human embryonic stem cells (hESC) have the capacity to differentiate in vivo and in vitro into cells from all three germ lineages. The aim of the present study was to investigate the effect of specific culture conditions on the differentiation of hESC into lung epithelial cells. METHODS: Undifferentiated hESC, grown on a porous membrane in hESC medium for four days, were switched to a differentiation medium for four days; this was followed by culture in air-liquid interface conditions during another 20 days. Expression of several lung markers was measured by immunohistochemistry and by quantitative real-time RT-PCR at four different time points throughout the differentiation and compared to appropriate controls. RESULTS: Expression of CC16 and NKX2.1 showed a 1,000- and 10,000- fold increase at day 10 of differentiation. Other lung markers such as SP-C and Aquaporin 5 had the highest expression after twenty days of culture, as well as two markers for ciliated cells, FOXJ1 and beta-tubulin IV. The results from qRT-PCR were confirmed by immunohistochemistry on paraffin-embedded samples. Antibodies against CC16, SP-A and SP-C were chosen as specific markers for Clara Cells and alveolar type II cells. The functionality was tested by measuring the secretion of CC16 in the medium using an enzyme immunoassay. CONCLUSION: These results suggest that by using our novel culture protocol hESC can be differentiated into the major cell types of lung epithelial tissue.

Regeneration of spermatogenesis by grafting testicular tissue or injecting testicular cells into the testes of sterile mice: a comparative study.

OBJECTIVE: To make a comparison between two different approaches-spermatogonial stem cell transplantation and intratesticular grafting, for preservation and reintroduction of spermatogonial stem cells. DESIGN: Prospective experimental study. SETTING: Academic medical center and teaching hospital. PATIENT(S): N/A. INTERVENTION(S): Intratesticular transplantation, histologic evaluation of testes. MAIN OUTCOME MEASURE(S): Testicular weight, amount of green fluorescence in the testis, and immunostaining for green fluorescent protein. RESULT(S): In a first experiment donor-derived spermatogenesis was found in 65% of the injected testes (41.8 +/- 72.2 mm) compared with 75% of the testes (122.1 +/- 45.6 mm) after tissue grafting. In the second series of experiments complete spermatogenesis was found in 75% of the testes after fresh grafting (93.8 +/- 21.8 mm) compared with 88% after frozen-thawed tissue grafting (84.8 +/- 45.6 mm). CONCLUSION(S): Both approaches show that spermatogonial stem cells can successfully be introduced to the testis resulting in spermatogenesis. Tissue grafting produced a larger mean donor colony length and there was no significant difference between colonization efficiency using either fresh or frozen-thawed grafts. In a future clinical setting, grafting would be a simple and efficient way for reintroducing stem cells to the testis.

Bone marrow stem cells transplanted to the testis of sterile mice do not differentiate into spermatogonial stem cells and have no protective effect on fertility.

Four months after transplanting bone marrow cells into the testis, no differentiation to spermatogonial stem cells was observed. Bone marrow transplantation had no protective effect on fertility after chemotherapy.

Spermatogonial survival in long-term human prepubertal xenografts.

Although childhood cancer treatments are yielding higher survival rates, sterility remains one of their major side effects. For prepubertal boys, there currently are no options to preserve fertility. Testicular tissue banking, together with subsequent grafting, may become a strategy in the future. In this study, prepubertal human testicular tissue was xenografted. Testicular tissue from two patients who had severe sickle-cell anemia and who needed to undergo chemotherapy and bone marrow transplantation was grafted onto the backs of six Swiss nude mice. Four months after grafting, spermatogonia could be observed by immunohistochemistry with MAGE-A4 antibodies, and Sertoli cells could be visualized by vimentin staining. Because both Sertoli cells and spermatogonia survived, tissue grafting may become a means for restoring future fertility in prepubertal male cancer patients.

Autologous spermatogonial stem cell transplantation in man: current obstacles for a future clinical application.

Fertility preservation is becoming an important issue in the management of the quality of life of prepubertal boys undergoing cancer treatment. At present, the only theoretical option for preservation of fertility in these boys is the preservation of the spermatogonial stem cells for autologous intratesticular stem cell transplantation. In animal models, this technique has shown promising results. However, before translation to the clinic, some major concerns should be evaluated. Improving the efficiency of the technique is one of the first goals for further research, besides evaluation of the safety of the clinical application. Also, the cryopreservation of the spermatogonial stem cells needs extra attention, since this first step will be crucial in the success of any clinical application. Another concern is the risk of malignant contamination of the testicular tissue in childhood cancer patients. Extensive research in this field and especially on the feasibility of decontaminating the testicular tissue will be inevitable. Another important, though overlooked, issue is the prevention of damage to the testicular niche cells. Finally, xenografting and in vitro proliferation/maturation of the spermatogonia should be studied as alternatives for the transplantation technique.

Cryosurvival and spermatogenesis after allografting prepubertal mouse tissue: comparison of two cryopreservation protocols.

Although childhood cancer treatments are yielding higher survival rates, sterility remains one of the major side effects. For prepubertal boys there are currently no options to preserve fertility. Testicular tissue banking together with subsequent grafting may become a possible strategy in the future. In the present study, we compared two cryopreservation protocols using prepubertal murine testicular tissue. Fresh and cryopreserved testicular tissue was grafted subcutaneously on the back of immune-deficient mice for at least 3 months. Prepubertal murine tissue recovered well after cryopreservation with both ethylene glycol (EG) and dimethylsulfoxide (DMSO). While in fresh murine allografts, spermatozoa were observed in 23% of the tubules; in both the DMSO and the EG groups, 32% of the seminiferous tubules contained spermatozoa. However, with DMSO the structure of the seminiferous tubules was better preserved.

Computer-assisted motility analysis of spermatozoa obtained after spermatogonial stem cell transplantation in the mouse.

OBJECTIVE: To study the motility characteristics of epididymal spermatozoa after spermatogonial stem cell transplantation. DESIGN: Testicular cells from fertile donor mice were transplanted to the testis of genetically sterile recipient mice. Three to nine months later, the epididymal spermatozoa were isolated and used for a computer-assisted sperm motility analysis. Spermatozoa from fertile adult mice were used as control. SETTING: Murine transplantation model in an academic research environment. ANIMAL(S): Donors, 6-day-old male C57Bl x WBRej F1 mice; acceptors, 4- to 6-week-old W/W(v) mice of the same genetic background. INTERVENTION(S): Two to 10 microL from a 30-million/mL testicular cell suspension was injected through the efferent duct in the rete testis. MAIN OUTCOME MEASURE(S): Vitality, concentration, motility, individual sperm movement, and hyperactivity of the spermatozoa. RESULT(S): Vitality was comparable between the two groups; the concentration, motility, and hyperactivity of posttransplantation spermatozoa were significantly reduced. The movement pattern of the individual spermatozoon was normal at the time of isolation but decreased more rapidly during in vitro culture, compared with the case in controls. This difference already had reached a significant level 3 hours after culture, which is comparable with the duration of an IVF procedure. CONCLUSION(S): The reduced fertilization rate after IVF can thus be explained by a lower number of motile spermatozoa and the faster decrease of the individual sperm movement parameters.

Evaluation of in vivo conception after testicular stem cell transplantation in a mouse model shows altered post-implantation development.

BACKGROUND: Apart from research applications, testicular stem cell transplantation (TSCT) may one day also have valuable clinical applications. Therefore, it is important to investigate whether this technique is a safe method to have progeny. This controlled study aims at evaluating the fetuses and the live born offspring obtained after TSCT in male mice. METHODS: Male mice were mated with wild-type (WT) females after TSCT to produce offspring. First, fetuses were evaluated on the 17th gestational day. The length, weight and morphological age were compared to those of control mouse fetuses. The live born offspring were then investigated for their reproductive potential over three generations. RESULTS: The litter sizes after TSCT were decreased compared to controls. Fetuses showed developmental retardation of a quarter of a day, but no major external abnormalities were observed. The live born pups were able to produce normal litter sizes, at least until the third generation. CONCLUSIONS: Transplanted animals are able to reproduce naturally. Although litter sizes are lower and development is retarded, no major morphological or procreative abnormalities were observed.

Spermatogonial survival after grafting human testicular tissue to immunodeficient mice.

BACKGROUND: The xenografting of pre-pubertal human testicular tissue to an immunodeficient mouse is a theoretical strategy for restoring fertility in childhood cancer patients, while circumventing the risk of malignant recurrence. This study aimed at comparing the grafting of pre-pubertal and adult murine testicular tissue, as well as that of human adult testicular tissue, to two immunodeficient recipients, i.e. Swiss Nude mice and SCID-NOD mice. MATERIALS AND METHODS: In this study, we evaluated the survival of pre-pubertal and adult murine testicular tissues, and that of adult human testicular tissue after subcutaneous grafting to immunodeficient mice. RESULTS: After allografting pre-pubertal testicular tissue pieces, meiotic cells were observed in 69.1% of the grafts, while complete spermatogenesis was observed in 30.9%. All grafts of adult murine testicular tissue and 59.5% of the adult human testicular grafts showed sclerosis. However, in 21.6% of the adult human testicular grafts, spermatogonia were still observed, with increasing sclerosis in time. No significant differences were observed between the two mouse models under evaluation. CONCLUSION: After xenografting human adult testicular tissue to a recipient mouse, spermatogonia were maintained over a period of >195 days. However, in order to prove xenografting as a method for external germ line storage, the transplants should have a more immature developmental stage. Moreover, not only the developmental status of the tissue at the time-point of grafting, but also the structural organisation of the seminiferous epithelium, might influence the development of the testicular tissue.

Preserving the reproductive potential of men and boys with cancer: current concepts and future prospects.

The introduction of ICSI has totally changed the reproductive prospects for boys and men who are treated for cancer. With post-pubertal boys and adult men, semen cryopreservation should be offered to every patient undergoing a cancer treatment since preservation of fertility cannot be guaranteed for an individual patient and treatment may shift to a more sterilizing regimen. In the ICSI era, all semen samples, even those containing only a few motile sperm, should be accepted for cryopreservation. Patients who are azoospermic at the time cancer is diagnosed may be offered testicular sperm extraction and cryopreservation of testicular tissue. With pre-pubertal boys, no prevention of sterility by sperm banking is possible since no active spermatogenesis is present. However, in the next decade, prevention of sterility in childhood cancer survivors will become a major challenge for reproductive medicine. In theory, testicular stem cell banking is the only way of preserving the future fertility of boys undergoing a sterilizing chemotherapy. In animal models, testicular stem cell transplantation has proved to be effective; however, it remains to be shown that this technique is clinically efficient as well, especially when frozen-thawed cells are to be transplanted. Malignancy recurrence prevention is an important prerequisite for any clinical application of testicular stem cell transplantation. Although still at the experimental stage, cryobanking of testicular tissue from pre-pubertal boys may now be considered an acceptable strategy.