Propagation of bovine spermatogonial stem cells in vitro

The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. A specialized medium and several growth factors were tested to study the in vitro behavior of bovine type A spermatogonia, a cell population that includes the SSCs and can be specifically stained for the lectin Dolichos biflorus agglutinin. During short-term culture (2 weeks), colonies appeared, the morphology of which varied with the specific growth factor(s) added. Whenever the stem cell medium was used, round structures reminiscent of sectioned seminiferous tubules appeared in the core of the colonies. Remarkably, these round structures always contained type A spermatogonia. When leukemia inhibitory factor (LIF), epidermal growth factor (EGF), or fibroblast growth factor 2 (FGF2) were added, specific effects on the numbers and arrangement of somatic cells were observed. However, the number of type A spermatogonia was significantly higher in cultures to which glial cell line-derived neurotrophic factor (GDNF) was added and highest when GDNF, LIF, EGF, and FGF2 were all present. The latter suggests that a proper stimulation of the somatic cells is necessary for optimal stimulation of the germ cells in culture. Somatic cells present in the colonies included Sertoli cells, peritubular myoid cells, and a few Leydig cells. A transplantation experiment, using nude mice, showed the presence of SSCs among the cultured cells and in addition strongly suggested a more than 10 000-fold increase in the number of SSCs after 30 days of culture. These results demonstrate that bovine SSC self-renew in our specialized bovine culture system and that this system can be used for the propagation of these cells.     

Proliferation and differentiation of spermatogonial stem cells

Spermatogonial stem cells (A(s) spermatogonia) are single cells that either renew themselves or produce A(pr) (paired) spermatogonia predestined to differentiate. In turn, the A(pr) divide into chains of A(al) (aligned) spermatogonia that also divide. The ratio between self-renewal and differentiation of the stem cells is regulated by glial cell line-derived neurotrophic factor produced by Sertoli cells, while the receptors are expressed in stem cells. A(s), A(pr) and A(al) spermatogonia proliferate during part of the epithelial cycle forming many A(al) spermatogonia. During epithelial stage VIII, almost all A(al) spermatogonia, few A(pr) and very few A(s) spermatogonia differentiate into A1 spermatogonia. A number of molecules are involved in this differentiation step including the stem cell factor-c-kit system, the Dazl RNA binding protein, cyclin D(2) and retinoic acid. There is no fine regulation of the density of spermatogonial stem cells and consequently, in some areas, many A1 and, in other areas, few A1 spermatogonia are formed. An equal density of spermatocytes is then obtained by the apoptosis of A2, A3 or A4 spermatogonia to remove the surplus cells. The Bcl-2 family members Bax and Bcl-x(L) are involved in this density regulation. Several mechanisms are available to cope with major or minor shortages in germ cell production. After severe cell loss, stem cell renewal is preferred above differentiation and the period of proliferation of A(s), A(pr) and A(al) spermatogonia is extended. Minor shortages are dealt with, at least in part, by less apoptosis among A2-A4 spermatogonia.     

Production of donor-derived sperm after spermatogonial stem cell transplantation in the dog

Spermatogonial stem cell transplantation (SSCT) offers unique approaches to investigate SSC and to manipulate the male germline. We report here the first successful performance of this technique in the dog, which is an important model of human diseases. First, we investigated an irradiation protocol to deplete endogenous male germ cells in recipient testes. Histologic examination confirmed >95% depletion of endogenous spermatogenesis, but retention of normal testis architecture. Then, 5-month-old recipient dogs (n=5) were focally irradiated on their testes prior to transplantation with mixed seminiferous tubule cells (fresh (n=2) or after 2 weeks of culture (n=3)). The dogs receiving cultured cells showed an immediate allergic response, which subsided quickly with palliative treatment. No such response was seen in the dogs receiving fresh cells, for which a different injection medium was used. Twelve months post-injection recipients were castrated and sperm was collected from epididymides. We performed microsatellite analysis comparing DNA from the epididymal sperm with genomic DNA from both the recipients and the donors. We used six markers to demonstrate the presence of donor alleles in the sperm from one recipient of fresh mixed tubule cells. No evidence of donor alleles was detected in sperm from the other recipients. Using quantitative PCR based on single nucleotide polymorphisms (SNPs), about 19.5% of sperm were shown to be donor derived in the recipient. Our results demonstrate the first successful completion of SSCT in the dog, an important step toward transgenesis through the male germline in this valuable biomedical model.     

Successful transplantation of bovine testicular cells to heterologous recipients

While heterologous germ cell transplantation was successful in pigs and goats, autologous transplantation alone has been reported to result in donor-derived spermatogenesis in cattle. The objective of this study was to investigate whether the transplantation of heterologous germ cells could result in colonization of recipient testes in cattle of different breeds. Testicular cells were isolated from 8 Bos taurus donor bull calves and then transferred into 15 Bos indicus-cross bull calves. All animals were prepubertal, donors were aged 5-7 months and recipients 5-11 months, and scrotal circumferences ranged from 15 to 22 cm. Single cell suspensions of donor testicular cells, prepared by enzymatic digestion, were labelled with fluorescent dyes PKH26 or CFDA-SE, before transfer into the rete testis of recipients under ultrasonographic guidance. To assess the longevity of colonization by donor cells, recipients were castrated 2-30 weeks after cell transfer. Donor cells were observed in 15/25 (60%) of the testes that received PKH26-labelled cells, whereas no CFDA-SE-positive cell was identified in any recipients. The maturity of the donors or recipients (measured by scrotal circumference) did not affect colonization potential. In freshly isolated tubules, clumps of PKH26-positive cells were observed, which indicated either cell division or extensive local colonization of specific areas of the tubules. In frozen sections, PKH26-positive cells were identified on the seminiferous tubule basement membrane, which indicated that these cells had successfully migrated from the tubule lumen and were likely to be spermatogonia. We conclude that PKH26 was more suitable for labelling donor testis cells and donor cells can be identified up to 6 months following transfer. These results indicate that allogeneic transplantation of testicular cells can occur between Bos taurus and Bos indicus cattle. Further studies will investigate functionality of transferred testicular cells.     

Reprogramming somatic cells into stem cells

Recent scientific achievements in cell and developmental biology have provided unprecedented opportunities for advances in biomedical research. The demonstration that fully differentiated cells can reverse their gene expression profile to that of a pluripotent cell, and the successful derivation and culture of human embryonic stem cells (ESCs) have fuelled hopes for applications in regenerative medicine. These advances have been put to public scrutiny raising legal, moral and ethical issues which have resulted in different levels of acceptance. Ethical issues concerning the use of cloned human embryos for the derivation of stem cells have stimulated the search for alternative methods for reversing differentiated cells into multi/pluripotent cells. In this article, we will review the present state of these reprogramming technologies and discuss their relative success. We also overview reprogramming events after somatic cell nuclear transfer (SCNT), as they may further instruct ex ovo strategies for cellular manipulation.     

Dose-response of RAG2-/-/gammac-/- mice to busulfan in preparation for spermatogonial transplantation

Practical applications of spermatogonial transplantation require good rates of colonization by the donor cells. Recipient testes are usually depleted of competing endogenous spermatogonia by administration of 32-44 mg busulfan kg(-1) body weight before transplantation. However, it is not clear that this is the optimum dose, especially for immunodeficient mice. In the present study, the response of adult RAG2(-/-)/gamma(c)(-/-) (RAG2) male mice to treatment with 10-50 mg busulfan kg(-1) body weight was determined in terms of mortality rates, testicular masses and histology, and colonization of seminiferous tubules by transplanted spermatogonia. Mortality increased from 0 to 50% at doses between 20 mg busulfan kg(-1) and 40 mg busulfan kg(-1), whereas the maximum effects on testicular mass and histology were observed at 20 mg busulfan kg(-1). Colonization of testes by genetically marked spermatogonia after treatment of mice with 20 mg busulfan kg(-1) was equivalent to rates previously reported in recipients treated with 32-44 mg busulfan kg(-1). Thus, 20 mg busulfan kg(-1) appears to be the optimum dose for preparing RAG2 mice for spermatogonial transplantation. However, because the steepness of the dose-response curves indicates that direct administration of busulfan is not ideal for this purpose, 15 mg busulfan kg(-1) was administered to pregnant females at various times between day 10.5 and day 16.5 of gestation to determine whether this would deplete the number of germ cells in male offspring. Although there were large variations in testicular mass and histology, no mortality was observed and administration of busulfan at day 10.5 or 12.5 after mating delayed initiation of spermatogenesis, indicating that prenatal administration of busulfan combined with neonatal transplantation might be an effective method for further increasing rates of colonization by donor spermatogonia.     

Germ cells from mouse and human embryonic stem cells

Mammalian gametes are derived from a founder population of primordial germ cells (PGCs) that are determined early in embryogenesis and set aside for unique development. Understanding the mechanisms of PGC determination and differentiation is important for elucidating causes of infertility and how endocrine disrupting chemicals may potentially increase susceptibility to congenital reproductive abnormalities and conditions such as testicular cancer in adulthood (testicular dysgenesis syndrome). Primordial germ cells are closely related to embryonic stem cells (ESCs) and embryonic germ (EG) cells and comparisons between these cell types are providing new information about pluripotency and epigenetic processes. Murine ESCs can differentiate to PGCs, gametes and even blastocysts - recently live mouse pups were born from sperm generated from mESCs. Although investigations are still preliminary, human embryonic stem cells (hESCs) apparently display a similar developmental capacity to generate PGCs and immature gametes. Exactly how such gamete-like cells are generated during stem cell culture remains unclear especially as in vitro conditions are ill-defined. The findings are discussed in relation to the mechanisms of human PGC and gamete development and the biotechnology of hESCs and hEG cells.     

Host-pathogen interactions in bovine mammary epithelial cells and HeLa cells by Staphylococcus aureus isolated from subclinical bovine mastitis

Staphylococcus aureus is a common pathogen that causes subclinical bovine mastitis due to several virulence factors. In this study, we analyzed S. aureus isolates collected from the milk of cows with subclinical mastitis that had 8 possible combinations of bap, icaA, and icaD genes, to determine their capacity to produce biofilm on biotic (bovine primary mammary epithelial cells and HeLa cells) and abiotic (polystyrene microplates) surfaces, and their ability to adhere to and invade these cells. We also characterized isolates for microbial surface components recognizing adhesive matrix molecules (MSCRAMM) and agr genes, and for their susceptibility to cefquinome sulfate in the presence of biofilm. All isolates adhered to and invaded both cell types, but invasion indexes were higher in bovine primary mammary epithelial cells. Using tryptic soy broth + 1% glucose on abiotic surfaces, 5 out of 8 isolates were biofilm producers, but only the bap⁺icaA⁺icaD⁺ isolate was positive in Dulbecco's Modified Eagle's medium. The production of biofilm on biotic surfaces occurred only with this isolate and only on HeLa cells, because the invasion index for bovine primary mammary epithelial cells was too high, making it impossible to use these cells in this assay. Of the 5 biofilm producers in tryptic soy broth + 1% glucose, 4 presented with the bap/fnbA/clfA/clfB/eno/fib/ebpS combination, and all were protected from cefquinome sulfate. We found no predominance of any agr group. The high invasive potential of S. aureus made it impossible to observe biofilm in bovine primary mammary epithelial cells, and we concluded that cells with lower invasion rates, such as HeLa cells, were more appropriate for this assay.     

Activation of immune cells in bovine mammary gland secretions by zymosan-treated bovine serum

Mastitis, caused by bacterial infection of the mammary gland, is a major disease of dairy cattle. The greatest risks of intramammary infection occur at the end of lactation and at the initiation of the next lactation when the cow calves. Treating serum with zymosan (yeast cell wall preparation) causes the complement to cleave, allowing this serum to serve as a source of complement fragment 5a (C5a), a potent chemoattractant and activator of the immune system. Our hypothesis was that intramammary infusion of zymosan-treated serum (ZTS) would recruit polymorphonuclear neutrophils (PMN) and generate prolonged activity in lymphocytes within the mammary gland. Ultimately this could help prevent bacterial infections in cows at dry-off and at the initiation of lactation. Two ipsilateral quarters of the mammary gland of each cow were infused with ZTS (12.5 mL/quarter), and 2 contralateral quarters were infused with saline in 8 cows shortly after lactation ended. Mammary secretions were collected periodically throughout the dry period and the first 2 wk of the next lactation. Activation status of lymphocytes and PMN in those secretions was assessed based on the intracellular presence or absence of IFN-γ and IL-8 as determined by flow cytometry. The ZTS infusion greatly increased PMN numbers in mammary secretions for the first week only. The percentage of IFN-γ positive lymphocytes and PMN, and the percentage of IL-8 positive PMN, exhibited a sustained increase in secretions from ZTS-treated quarters through the first 2 wk of lactation. The ZTS can stimulate PMN and lymphocyte-mediated immune defense mechanisms in the mammary gland, which may provide a useful means of preventing new intramammary infections during the dry period as well as at the initiation of lactation.     

Culture systems for pluripotent stem cells

Pluripotent stem cells have the capacity to self renew and to differentiate to cells of the three somatic germ layers that comprise an organism. Embryonic stem cells are the most studied pluripotent stem cells. Pluripotent stem cells have also been derived from adult tissues. Both embryonic and adult stem cells represent valuable sources of cells for applications in cell therapy, drug screening and tissue engineering. While expanding stem cells in culture, it is critical to maintain their self-renewal and differentiation capacity. In generating particular cell types for specific applications, it is important to direct their differentiation to the desired lineage. Challenges in expansion of undifferentiated stem cells for clinical applications include the removal of feeder layers and non-defined components in the culture medium. Our limited basic knowledge on the requirements for maintaining pluripotency of adult pluripotent stem cells and the lack of appropriate markers associated with pluripotency hinders the progress toward their wide spread application. In vitro differentiation of stem cells usually produces a mixed population of different cell lineages with the desired cell type present only at a small proportion. Use of growth factors that promote differentiation, expansion or survival of specific cell types is key in controlling the differentiation towards specific cell lineages. A variety of bioreactors for cell cultivation exist and can be readily adapted for stem cell cultivation and differentiation. They provide a well-controlled environment for studying the process of stem cell propagation and differentiation. Their wide use will facilitate the development of processes for stem cell application.     

Production of matrix metalloproteinases by cultured bovine theca and granulosa cells

Matrix metalloproteinases (MMPs) degrade the proteinaceous components of the extracellular matrix and are presumably essential for follicular growth culminating in ovulation or atresia. The objectives of this study were to characterize the gelatinolytic and caseinolytic MMPs secreted by cultured bovine thecal and granulosal cells and to determine the effect of luteinizing hormone (LH) on MMP secretion. Thecal and granulosal cells were collected from small bovine follicles (<5 mm) on day 2 or 5 of the estrous cycle (day 0=estrus). A serum-free culture system was utilized in which bovine thecal and granulosal cells do not spontaneously luteinize, but produce androstenedione and estradiol in response to physiological concentrations of LH and follicle-stimulating hormone (FSH) respectively. The effect of LH (0, 1 or 100 ng/ml) on MMP production was determined in conditioned media collected every 48 h for 144 h. MMPs were detected by gelatin and casein zymography and MMP activity was quantified by image analysis. Thecal and granulosal cell conditioned media contained MMPs that had a relative molecular size (Mr) ranging from 53,000 to 200,000 and addition of 1,10 phenanthroline (MMP inhibitor) blocked gelatinolytic and caseinolytic activity. Patterns of gelatinolytic activity in thecal and granulosal cell conditioned media differed over time with the Mr 62,000 and 83,000 MMPs being increased (P <0.05) and the Mr 53,000 MMP being decreased (P <0.05) at 96 h of culture. LH (1 or 100 ng/ml) increased (P <0.05) gelatinolytic activity of the Mr 53,000 and 62,000 gelatinases within thecal cell conditioned media but not granulosal cell conditioned media. The Mr 62,000 and 83,000 gelatinolytic activities corresponded to the active forms of gelatinase A (Mr 62,000) and B (Mr, 83,000) and gelatinase A was detected in thecal cell conditioned media by Western blot analysis. Caseinolytic activity (Mr 83,000) was detected in both thecal and granulosal cell conditioned media and increased from 48 to 96 h. In summary, thecal and granulosal cells secrete gelatinolytic and caseinolytic MMPs and thecal cell production of gelatinase A was stimulated by LH.     

Comparison of efficiency of terminal differentiation of oligodendrocytes from induced pluripotent stem cells versus embryonic stem cells in vitro

Oligodendrocytes are the myelinating cells of the central nervous system (CNS), and defects in these cells can result in the loss of CNS functions. Although oligodendrocyte progenitor cells transplantation therapy is an effective cure for such symptoms, there is no readily available source of these cells. Recent studies have described the generation of induced pluripotent stem cells (iPS cells) from somatic cells, leading to anticipation of this technique as a novel therapeutic tool in regenerative medicine. In this study, we evaluated the ability of iPS cells derived from mouse embryonic fibroblasts to differentiate into oligodendrocytes and compared this with the differential ability of mouse embryonic stem cells (ES cells). Experiments using an in vitro oligodendrocyte differentiation protocol that was optimized to ES cells demonstrated that 2.3% of iPS cells differentiated into O4⁺ oligodendrocytes compared with 24.0% of ES cells. However, the rate of induction of A2B5⁺ oligodendrocyte precursor cell (OPC) was similar for both iPS-derived cells and ES-derived cells (14.1% and 12.6%, respectively). These findings suggest that some intracellular factors in iPS cells inhibit the terminal differentiation of oligodendrocytes from the OPC stage.     

Culture of mammary epithelial cells from bovine milk.

Mammary epithelial cells are exfoliated into cows' milk and comprise a component of the somatic cells enumerated for milk quality control. This paper described the finding that these epithelial cells can be recovered from milk and grown in culture. The cultured cells were characterized for growth rate and growth potential, morphology by light and electron microscopy, presence of esterases and cytokeratins, and sensitivity to storage at 4 degrees C. Cultured mammary epithelial cells from milk may be useful to dairy scientists and mammary gland biologists.     

Derivation, growth and applications of human embryonic stem cells

Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass cells of blastocysts with the potential to maintain an undifferentiated state indefinitely. Fully characterised hES cell lines express typical stem cell markers, possess high levels of telomerase activity, show normal karyotype and have the potential to differentiate into numerous cell types under in vitro and in vivo conditions. Therefore, hES cells are potentially valuable for the development of cell transplantation therapies for the treatment of various human diseases. However, there are a number of factors which may limit the medical application of hES cells: (a) continuous culture of hES cells in an undifferentiated state requires the presence of feeder layers and animal-based ingredients which incurs a risk of cross-transfer of pathogens; (b) hES cells demonstrate high genomic instability and non-predictable differentiation after long-term growth; and (c) differentiated hES cells express molecules which could cause immune rejection. In this review we summarise recent progress in the derivation and growth of undifferentiated hES cells and their differentiated progeny, and the problems associated with these techniques. We also examine the potential use of the therapeutic cloning technique to derive isogenic hES cells.     

Cultured meat from stem cells: challenges and prospects

As one of the alternatives for livestock meat production, in vitro culturing of meat is currently studied. The generation of bio-artificial muscles from satellite cells has been ongoing for about 15 years, but has never been used for generation of meat, while it already is a great source of animal protein. In order to serve as a credible alternative to livestock meat, lab or factory grown meat should be efficiently produced and should mimic meat in all of its physical sensations, such as visual appearance, smell, texture and of course, taste. This is a formidable challenge even though all the technologies to create skeletal muscle and fat tissue have been developed and tested. The efficient culture of meat will primarily depend on culture conditions such as the source of medium and its composition. Protein synthesis by cultured skeletal muscle cells should further be maximized by finding the optimal combination of biochemical and physical conditions for the cells. Many of these variables are known, but their interactions are numerous and need to be mapped. This involves a systematic, if not systems, approach. Given the urgency of the problems that the meat industry is facing, this endeavor is worth undertaking. As an additional benefit, culturing meat may provide opportunities for production of novel and healthier products.     

Quantification and immunoglobulin classification of plasma cells in nonlactating bovine mammary tissue

Plasma cell populations in bovine mammary tissue were examined during involution using electron microscopic and immunohistochemical techniques. Biopsies were taken from each quarter of five Jersey cows at weekly intervals beginning at drying off through parturition. Ultrastructural examination of stromal plasma cells revealed rough endoplasmic reticulum cisternae engorged with flocculent material, indicative of antibody synthesis. Plasma cells were observed proximal to alveolar epithelial cells. This association may facilitate transport of antibody through epithelium and into milk. Immunoglobulin-producing plasma cell numbers increased gradually from drying off, reached peak concentrations 2 wk prepartum, and dropped significantly during the last 2 wk of gestation. Plasma cells producing immunoglobulin G1 and G2 were the most numerous types observed during the nonlactating period followed by cells producing immunoglobulin M and immunoglobulin A. Plasma cells producing immunoglobulin were more numerous during the last 2 wk of gestation and in tissue infected with minor pathogens than in uninfected quarters. Exposure to minor pathogens may have enhanced sensitized B-lymphocyte proliferation into antibody producing plasma cells through antigenic stimulation. Results of plasma cell distribution during the nonlactating period in bovine mammary tissue indicate times when local immunostimulation of B-lymphocytes may be most effective in enhancing immunity to intramammary infection.     

Cell microarray for screening feeder cells for differentiation of embryonic stem cells

Microarrays are currently recognized as one of major tools in the assessment of gene expression via cDNA or RNA analysis and are now accepted as a powerful experimental tool for high-throughput screening of a large number of samples, such as cDNA and siRNAs. In this study, we examined the potential of the microarray methodology for high-throughput screening of candidate cells as feeder cells which effectively differentiate embryonic stem (ES) cells to the specific lineage. Cell arrays were prepared by applying three kinds of cells, PA6, human umbilical vein endothelial, and COS-1 cells, to circular spots, 2 mm in diameter, on a glass plate, followed by the application of mouse ES cells to the cell microarray. After 8 d in culture, TuJ1 (neuron-specific class III beta-tubulin) immunocytochemical staining clearly demonstrated that only PA6 cell spots had the capability to induce ES cells to neuronal differentiation. Although this is a model experiment, these findings clearly indicate that the cell microarray will become a powerful tool for high-throughput screening large numbers of candidate feeder cells for specific differentiation.