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Antibodies are among the most common tools in basic science and clinical research. However, an increasing number of studies have shown that not all antibodies are specific, leading to a growing issue of experimental irreproducibility. This is in part due to the cross-reactivity between antibodies and off-target proteins, as well as the variability between different antibody batches. These unspecific antibodies result in wasted time and resources and compromise the advancement of science. In response to this, Abcam launched a knockout (KO) validation program in 2015 as one of its antibody quality control protocols. This initiative uses KO cells lines made possible through a partnership with Horizon Discovery. Target genes are mutated via CRISPR-Cas9 within a haploid cell line, which results in a frameshift and a complete loss of gene expression. These KO cell lines function as true negative controls that can be used at a large scale to confirm antibody specificity to the protein of interest. We have KO-validated hundreds of antibodies, including several that are relevant to immunology and immuno-oncology research. Here we present the data related to key targets validated in western blot, flow cytometry and immunocytochemistry. The antibodies have been tested in KO and wild-type cells to confirm their specificity. By providing researchers with reliable and specific antibodies that work first time, we hope to minimize wasted resources and improve antibody reproducibility.

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.

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.

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.

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.

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.

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.

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 × WBRej F1 mice; acceptors, 4- to 6-week-old W/W v mice of the same genetic background. Intervention(s) Two to 10 μL 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.

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.

Transplantation of frozen-thawed testicular cell suspensions.

Embryo development after ICSI with spermatozoa obtained after stem cell transplantation. Goossens E., Frederickx V., De Block G., Van Steirteghem A., Tournaye H. Centre for Reproductive Medicine and Research Laboratories for Reproductive Medicine, University Hospital and Medical School, Dutch-speaking Brussels Free University (Vrije Universiteit Brussel), Laarbeeklaan 101, B-1090 Brussels, Belgium. ellen.goossens@az.vub.ac.be Introduction The stem cell transplantation technique, introduced by Brinster and colleagues (1), has proven its efficacy in the mouse. This technique may also be useful in clinical applications, i.g. for prepubertal boys who need to undergo cancer therapy. The stem cells of one testis could be frozen and transplanted in the remaining testis once the patient is cured. When these boys become adults and want to have children, they may not produce enough sperm for natural conception and may need assisted reproduction. This study aimed at evaluating the efficiency of intracytoplasmic sperm injection (ICSI) with sperm cells obtained after stem cell transplantation. Materials & Methods A testicular cell suspension, obtained from six-day old F1 hybrid (C57Bl/6 x Cba) males, was transplanted in the testis of sterile W/Wv-mice. Three months after transplantation motile spermatozoa were retrieved from the epididymis and injected in metaphase II (MII)-oocytes from B6D2F1 (C57Bl/6 x Dba/2) females. We used the conventional ICSI method, as described by Suzuki and Yanagimachi (2). After 24h the oocytes were checked for survival and fertilisation. Every following day, the embryonic development was evaluated. Epididymal sperm cells of fertile adult males were injected in MII-oocytes from B6D2F1 (C57Bl/6 x Dba/2) females and served as control. The Fisher-exact test was used for statistical analysis of the results. Results A testicular cell suspension was successfully transplanted in 26 testes of sixteen W/Wv-mice. After three months spermatozoa were found in eleven (42%) testes. Motile epididymal sperm cells from these mice were injected in 187 oocytes. Seventy (37%) survived the injection and 26 (37%) were fertilised. Eleven oocytes were arrested at the two-cell stage, eleven oocytes developed into the four-cell stage, two reached morula-stage and one (4%) became blastocyst (Table 1 and 2). Some fertilised oocytes had an abnormal morphology (fig. 1 and 2). The results for the control mice were as follows: from the 188 oocytes injected 81 (43%) survived injection and 30 (37%) were fertilised. Sixteen oocytes showed a two-cell block, five oocytes reached morula-stage and nine (30%) developed into blastocysts. Discussion Sperm cells obtained after testicular stem cell transplantation are able to fertilise oocytes by ICSI. Nevertheless, the developing embryos have an abnormal morphology and are often arrested in an early embryonic stage. References 1. Brinster, R. L. and Zimmerman, J.W. (1994) Proc. Natl. Acad. Sci. 91, 11298-11302. 2. Suzuki, K. and Yanagimachi, R. (1997) Zygote 5, 111-116.

Letter to Editor: Reply to: Isolation of germ cells from leukemic cells. Fujita K, Tsujimura A, Okuyama A. Hum Reprod. 2007 Oct;22(10):2796-7; author reply 2797-8. Epub 2007 Aug 28.

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.

Introduction: The testicular stem cell transplantation technique has become an established research model in the mouse. This technique may also become useful for clinical applications. Therefore, it is necessary to investigate whether spermatozoa obtained after testicular stem cell transplantation retain their full functional capacity and whether they are able to produce normally developing embryos. This study aimed at evaluating the fertilising and developmental abilities of spermatozoa obtained after stem cell transplantation. Materials/methods: First, transplanted male mice were mated with females in order to evaluate in-vivo conception. Subsequently, functionality of spermatozoa obtained after testicular germ cell transplantation was investigated by performing both in-vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI). Results: After in-vivo conception we found that in the control group 90% of the mice with a copulating plug became pregnant. In the experimental group only 35% of the mice with a copulating plug became pregnant (p=0.006). After IVF, fertilisation and blastocyst developmental rates were significantly lower in the transplanted group (p

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 β-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.