Stem cells are defined as cells able to both extensively self-renew and differentiate into progenitors. Research data show that more resistant stem cells than common cancer cells exist in cancer patients.To identify unrecognized differences between cancer stem cells and cancer cells might be able to develope effective classification,diagnose and treat ment for cancer.

Stem Cells,Cancer and Cancer Stem Cells

Five structural features in mRNAs have been found to contribute to the fidelity and efficiency of initiation by eukaryotic ribosomes. Scrutiny of vertebrate cDNA sequences in light of these criteria reveals a set of transcripts--encoding oncoproteins, growth factors, transcription factors, and other regulatory proteins--that seem designed to be translated poorly. Thus, throttling at the level of translation may be a critical component of gene regulation in vertebrates. An alternative interpretation is that some (perhaps many) cDNAs with encumbered 5' noncoding sequences represent mRNA precursors, which would imply extensive regulation at a posttranscriptional step that precedes translation.

https://rupress.org/jcb/article-pdf/115/4/887/1062268/887.pdf

An analysis of vertebrate mRNA sequences: intimations of translational control.

The concept that stem cells are controlled by particular microenvironments known as 'niches' has been widely invoked. But niches have remained largely a theoretical construct because of the difficulty of identifying and manipulating individual stem cells and their surroundings. Technical advances now make it possible to characterize small zones that maintain and control stem cell activity in several organs, including gonads, skin and gut. These studies are beginning to unify our understanding of stem cell regulation at the cellular and molecular levels, and promise to advance efforts to use stem cells therapeutically.

Stem cells find their niche

Male reproductive health has deteriorated in many countries during the last few decades. In the 1990s, declining semen quality has been reported from Belgium, Denmark, France, and Great Britain. The incidence of testicular cancer has increased during the same time incidences of hypospadias and cryptorchidism also appear to be increasing. Similar reproductive problems occur in many wildlife species. There are marked geographic differences in the prevalence of male reproductive disorders. While the reasons for these differences are currently unknown, both clinical and laboratory research suggest that the adverse changes may be inter-related and have a common origin in fetal life or childhood. Exposure of the male fetus to supranormal levels of estrogens, such as diethlylstilbestrol, can result in the above-mentioned reproductive defects. The growing number of reports demonstrating that common environmental contaminants and natural factors possess estrogenic activity presents the working hypothesis that the adverse trends in male reproductive health may be, at least in part, associated with exposure to estrogenic or other hormonally active (e.g., antiandrogenic) environmental chemicals during fetal and childhood development. An extensive research program is needed to understand the extent of the problem, its underlying etiology, and the development of a strategy for prevention and intervention.

Male reproductive health and environmental xenoestrogens

In the adult male, a population of diploid stem-cell spermatogonia continuously undergoes self-renewal and produces progeny cells, which initiate the complex process of cellular differentiation that results in mature spermatozoa. We report here that stem cells isolated from testes of donor male mice will repopulate sterile testes when injected into seminiferous tubules. Donor cell spermatogenesis in recipient testes showed normal morpholigical characteristics and produced mature spermatozoa. This methodology, besides opening new avenues of basic research into spermatogenesis and stem-cell self-renewal, may prove useful as a tool for biomedical science and biotechnology.

https://www.pnas.org/content/pnas/91/24/11298.full.pdf

Spermatogenesis following male germ-cell transplantation

Spermatogenesis is a complex, highly organized, very efficient process that is based upon the capacity of stem cell spermatogonia simultaneously to undergo self-renewal and to provide progeny that differentiate into mature spermatozoa. We report here that testis-derived cells transplanted into the testis of an infertile mouse will colonize seminiferous tubules and initiate spermatogenesis in > 70% of recipients. Testis-derived cells from newborn mice were less effective in colonizing recipient testes than cells from 5- to 15- or 21- to 28-day-old mice. Increasing the number of Sertoli cells in the donor cell population did not increase the efficiency of colonization. Unmodified embryonic stem cells were not able to substitute for testis-derived cells in colonizing testes but instead formed tumors in syngeneic as well as nonsyngeneic hosts. Finally, with recipients that maintained endogenous spermatogenesis, testis cell transplantation yielded mice in which up to 80% of progeny were sired by donor-derived spermatozoa. The technique of spermatogonial cell transplantation should provide a means to generate germline modifications in a variety of species following development of spermatogonial culture techniques and should have additional applications in biology, medicine, and agriculture.

https://www.pnas.org/content/pnas/91/24/11303.full.pdf

Germline transmission of donor haplotype following spermatogonial transplantation.

Spermatogonial stem cells (SSCs) self-renew and produce large numbers of committed progenitors that are destined to differentiate into spermatozoa throughout life. However, the growth factors essential for self-renewal of SSCs remain unclear. In this study, a serum-free culture system and a transplantation assay for SSCs were used to identify exogenous soluble factors that promote proliferation of SSCs. Mouse pup testis cells were enriched for SSCs by selection with an anti-Thy-1 antibody and cultured on STO (SIM mouse embryo-derived thioguanine and ouabain resistant) feeders in a serum-free defined medium. In the presence of glial cell line-derived neurotrophic factor (GDNF), SSCs from DBA/2J strain mice formed densely packed clumps of cells and continuously proliferated. However, other strains of mice required the addition of soluble GDNF-family receptor α-1 and basic fibroblast growth factor to support replication. The functional transplantation assay proved that the clump-forming cells are indeed SSCs. Thus, GDNF-induced cell signaling plays a central role in SSC self-renewal. The number of SSCs in culture doubled every 5.6 days, and the clump-forming cells strongly expressed Oct-4. Under these conditions, SSCs proliferated over 6 months, reconstituted long-term spermatogenesis after transplantation into recipient testes, and restored fertility to infertile recipients. The identification of exogenous factors that allow continuous proliferation of SSCs in vitro establishes the foundation to study the basic biology of SSCs and makes possible germ-line modification by sophisticated technologies. Moreover, the ability to recover, culture indefinitely, and transplant SSCs will make the germ-line of individual males available for periods extending beyond a normal lifetime.

https://www.pnas.org/content/pnas/101/47/16489.full.pdf

Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells

In the adult male, germ cell differentiation takes place in the seminiferous tubules of the testis by a complex, highly organized and very efficient process. A population of diploid stem-cell spermatogonia that lie on the basement membrane of the tubule continuously undergoes self-renewal and produces progeny cells, which initiate the process of cellular differentiation to generate mature spermatozoa. Each testis contains many seminiferous tubules, which are connected at both ends to a collecting system called the rete testis. The mature spermatozoa pass from the tubules into the rete and are then carried through efferent ducts to the epididymis for final maturation before they are ready to fertilize an egg. In previous studies, we have demonstrated that donor testis cells collected from a fertile mouse are able to generate spermatogenesis when transplanted to the seminiferous tubules of an infertile male. The spermatozoa produced by the recipient from the donor-derived spermatogonial stem cells are able to fertilize eggs and produce progeny carrying the donor male haplotype. Furthermore, donor testis stem cells from a rat will generate normal rat spermatozoa following transplantation to a mouse testis. The spermatogonial transplantation technique is clearly valuable and applicable to many species, but it is difficult. Therefore, several procedures to introduce donor cells into the seminiferous tubules of a recipient have been developed using the mouse as a model, and they are described here in detail. The results indicate that microinjection of cell suspensions into the seminiferous tubules, efferent ducts or rete testis are equally effective in generating donor cell-derived spermatogenesis in recipients. Each approach is likely to be useful for different experimental purposes in a variety of species.

/pdf/transplantation-of-testis-germinal-cells-into-mouse-3nqdxwa7oq.pdf

Transplantation of testis germinal cells into mouse seminiferous tubules.

Although spermatogenesis is essential for reproduction, little is known about spermatogonial stem cells. These cells provide the basis for spermatogenesis throughout adult life by undergoing self-renewal and by providing progeny that differentiate into spermatozoa. A major impediment to our understanding of the biology of these stem cells is the inability to distinguish them from spermatogonia that are committed to differentiation. We made use of the known association of stem cells with basement membranes and our spermatogonial transplantation assay system to identify specific molecular markers on the stem cell surface. Selection of mouse testis cells with anti-β1- or anti-α6-integrin antibody, but not anti-c-kit antibody, produced cell populations with a significantly enhanced ability to colonize recipient testes and generate donor cell-derived spermatogenesis. We demonstrate spermatogonial stem cell-associated antigens by using an assay system based on biological function. Furthermore, the presence of surface integrins on spermatogonial stem cells suggests that these cells share elements of a common molecular machinery with stem cells in other tissues.

https://www.pnas.org/content/pnas/96/10/5504.full.pdf

β1- and α6-integrin are surface markers on mouse spermatogonial stem cells

In a previous study we showed that genomic constructs were expressed more efficiently in transgenic mice than constructs that were identical except for the lack of introns. Using the mouse metallothionein promoter-rat growth hormone gene construct as a model, we show that the first intron of the rat growth hormone gene is essential for high-level expression, whereas the other three introns are less effective. Several heterologous introns placed 3' of the coding region of an intronless rat growth hormone gene are also ineffective. However, insertion of some heterologous introns between the metallothionein promoter and the growth hormone gene improves expression. To determine whether addition of heterologous introns would provide a general strategy for improving expression, we have tested them in conjunction with other intronless genes and with different promoters.

https://www.pnas.org/content/pnas/88/2/478.full.pdf

Mary R. Avarbock

Papers

Germline transmission of donor haplotype following spermatogonial transplantation.

Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells

Transplantation of testis germinal cells into mouse seminiferous tubules.

β1- and α6-integrin are surface markers on mouse spermatogonial stem cells

Heterologous introns can enhance expression of transgenes in mice.