Gert De Block

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

BackgroundHuman 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.MethodsUndifferentiated 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.ResultsExpression 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.ConclusionThese results suggest that by using our novel culture protocol hESC can be differentiated into the major cell types of lung epithelial tissue.

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.

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.

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.