Developing a clinical-grade cryopreservation protocol for human testicular tissue and cells.

Recent work in preservation of female fertility as well as new information on the nature of spermatogonial stem cells has prompted an investigation into the possibility of an effective clinical-grade procedure for the cryopreservation of testicular cells and/or tissue. Clinical-grade reagents, validated equipment, and protocols consistent with cGTP/cGMP standards were used in developing a procedure suitable for the safe and effective cryopreservation of human testicular cells and tissues. These procedures were designed to be compliant with the relevant FDA regulations. The procedure proved to effectively cryopreserve both testicular cells and tissue. The cryopreservation of testicular tissue was comparable in most aspects we measured to the cryopreservation of isolated cells, except that the viability of the cells from cryopreserved testicular tissue was found to be significantly higher. On the other hand, cryopreservation of cells is preferred for cell analysis, quality control, and sterility testing. This study demonstrates that testicular tissue and cells from sexual reassignment patients can be successfully cryopreserved with a clinical-grade procedure and important cell populations are not only preserved but also enriched by the process. Further studies will determine whether these findings from hormone-treated patients can be generalized to other patients.

Identification and characterization of repopulating spermatogonial stem cells from the adult human testis.

BACKGROUND: This study was conducted to identify and characterize repopulating spermatogonial stem cells (SSCs) in the adult human testes. METHODS: Testes biopsies from obstructive azoospermic patients and normal segments of human testicular tissue were used. Flow cytometry, real-time PCR and immunohistochemical analysis were performed. Purified human spermatogonia were transplanted into busulfan-treated recipient mouse testes and integrated cells were detected by human nuclear protein antibody co-localized with stem cell and germ cell markers. RESULTS: Testicular biopsies collected from obstructive azoospermic men showed similar morphology and distribution of markers to the normal human testes. Flow cytometry showed distinct populations of stage-specific embryonic antigen-4 (SSEA-4), CD49f and CD90 positive cells in the adult human testes. SSEA-4 (+) cells showed high expression levels of SSC-specific genes and high levels of telomerase activity. Extensive colonization of human cells in the mouse testes indicates the presence of highly enriched populations of SSCs in the SSEA-4 (+) sorted cells. All the HNP (+) cells in the mouse testes were positive for germ cell marker dead box mRNA helicase and only half of them were dimly positive for c-kit. In addition, subpopulations of human spermatogonia that colonized mouse testes were positively stained for CD49f, GPR-125, Nanog and Oct-4 indicating the existence of population of cells among human spermatogonia with SSC and pluripotent characteristics. CONCLUSIONS: This study clearly demonstrates that repopulating human SSCs have phenotypic characteristics of SSEA-4(+), CD49f(+), GPR-125(+)and c-Kit (neg/low). The results have direct implications for enrichment of human spermatogonia for further culture and germ cell differentiation studies.

Differentiation potential of germ line stem cells derived from the postnatal mouse ovary.

General belief in reproductive biology is that in most mammals female germ line stem cells are differentiated to primary oocytes during fetal development and oogenesis starts from a pool of primordial follicles after birth. This idea has been challenged previously by using follicle kinetics studies and demonstration of mitotically active germ cells in the postnatal mouse ovary (Johnson et al., 2004; Kerr et al., 2006; Zhang et al., 2008). However, the existence of a population of self-renewing ovarian germ line stem cells in postnatal mammals is still controversial (Eggan et al., 2006; Telfer et al., 2005; Gosden, 2004). Recently, production of offspring from a germ line stem cell line derived from the neonatal mouse ovary was reported (Zou et al., 2009). This report strongly supports the existence of germ line stem cells and their ability to expand in vitro. Recently, using a transgenic mouse model in which GFP is expressed under a germ cell-specific Oct-4 promoter, we isolated and generated multipotent cell lines from male germ line stem cells (Izadyar et al., 2008). Using the same strategy we isolated and derived cell lines from postnatal mouse ovary. Interestingly, ovarian germ line stem cells expanded in the same culture conditions as the male suggesting that they have similar requirements for their self-renewal. After 1 year of culture and many passages, ovarian germ line stem cells maintained their characteristics and telomerase activity, expressed germ cell and stem cell markers and revealed normal karyotype. As standard protocol for differentiation induction, these cells were aggregated and their ability to form embryoid bodies (EBs) was investigated. EBs generated in the presence of growth factors showed classical morphology and expressed specific markers for three germ layers. However, in the absence of growth promoting factors EBs were smaller and large cells with the morphological and molecular characteristics of oocytes were formed. This study shows the existence of a population of germ line stem cell in postnatal mouse ovary with multipotent characteristics.

Establishment of cell lines from mouse embryos with early embryonic lethality.

It is often difficult to determine molecular mechanisms leading to early embryonic lethality of genetically modified mice due to lack of cells for further analyses. The authors describe here establishment of mouse embryonic fibroblast (MEF) cell lines from gastrulation stage embryos. In this example, using a combination of in vivo and in vitro techniques, the authors successfully generated MEF cell lines that lack both fibronectin (FN) and focal adhesion kinase (FAK).

Generation of multipotent cell lines from a distinct population of male germ line stem cells.

Spermatogonial stem cells (SSCs) maintain spermatogenesis by self-renewal and generation of spermatogonia committed to differentiation. Under certain in vitro conditions, SSCs from both neonatal and adult mouse testis can reportedly generate multipotent germ cell (mGC) lines that have characteristics and differentiation potential similar to embryonic stem (ES) cells. However, mGCs generated in different laboratories showed different germ cell characteristics, i.e., some retain their SSC properties and some have lost them completely. This raises an important question: whether mGC lines have been generated from different subpopulations in the mouse testes. To unambiguously identify and track germ line stem cells, we utilized a transgenic mouse model expressing green fluorescence protein under the control of a germ cell-specific Pou5f1 (Oct4) promoter. We found two distinct populations among the germ line stem cells with regard to their expression of transcription factor Pou5f1 and c-Kit receptor. Only the POU5F1+/c-Kit+ subset of mouse germ line stem cells, when isolated from either neonatal or adult testes and cultured in a complex mixture of growth factors, generates cell lines that express pluripotent ES markers, i.e., Pou5f1, Nanog, Sox2, Rex1, Dppa5, SSEA-1, and alkaline phosphatase, exhibit high telomerase activity, and differentiate into multiple lineages, including beating cardiomyocytes, neural cells, and chondrocytes. These data clearly show the existence of two distinct populations within germ line stem cells: one destined to become SSC and the other with the ability to generate multipotent cell lines with some pluripotent characteristics. These findings raise interesting questions about the relativity of pluripotency and the plasticity of germ line stem cells.

Nuclear FAK promotes cell proliferation and survival through FERM-enhanced p53 degradation.

FAK is known as an integrin- and growth factor-associated tyrosine kinase promoting cell motility. Here we show that, during mouse development, FAK inactivation results in p53- and p21-dependent mesodermal cell growth arrest. Reconstitution of primary FAK-/-p21-/- fibroblasts revealed that FAK, in a kinase-independent manner, facilitates p53 turnover via enhanced Mdm2-dependent p53 ubiquitination. p53 inactivation by FAK required FAK FERM F1 lobe binding to p53, FERM F2 lobe-mediated nuclear localization, and FERM F3 lobe for connections to Mdm2 and proteasomal degradation. Staurosporine or loss of cell adhesion enhanced FERM-dependent FAK nuclear accumulation. In primary human cells, FAK knockdown raised p53-p21 levels and slowed cell proliferation but did not cause apoptosis. Notably, FAK knockdown plus cisplatin triggered p53-dependent cell apoptosis, which was rescued by either full-length FAK or FAK FERM re-expression. These studies define a scaffolding role for nuclear FAK in facilitating cell survival through enhanced p53 degradation under conditions of cellular stress.

FAK promotes organization of fibronectin matrix and fibrillar adhesions.

Targeted disruption of the focal adhesion kinase (FAK) gene in mice is lethal at embryonic day 8.5 (E8.5). Vascular defects in FAK-/- mice result from the inability of FAK-deficient endothelial cells to organize themselves into vascular network. We found that, although fibronectin (FN) levels were similar, its organization was less fibrillar in both FAK-/- endothelial cells and mesoderm of E8.5 FAK-/- embryos, as well as in mouse embryonic fibroblasts isolated from mutant embryos. FAK catalytic activity, proline-rich domains, and location in focal contacts were all required for proper allocation and patterning of FN matrix. Cells lacking FAK in focal adhesions fail to translocate supramolecular complexes of integrin-bound FN and focal adhesion proteins along actin filaments to form mature fibrillar adhesions. Taken together, our data suggest that proper FN allocation and organization are dependent on FAK-mediated remodeling of focal adhesions.