Culture of spermatogonial stem cells and use of surrogate sires as a breeding technology to propagate superior genetics in livestock production: A systematic review

Background and Aim: Spermatogonial stem cells (SSCs) have previously been isolated from animals' testes, cultured in vitro, and successfully transplanted into compatible recipients. The SSC unique characteristic has potential for exploitation as a reproductive tool and this can be achieved through SSC intratesticular transplantation to surrogate sires. Here, we aimed at comprehensively analyzing published data on in vitro maintenance of SSC isolated from the testes of livestock animals and their applications. Materials and Methods: The literature search was performed in PubMed, Science Direct, and Google Scholar electronic databases. Data screening was conducted using Rayyan Intelligent Systematic Review software (https://www.rayyan.ai/). Duplicate papers were excluded from the study. Abstracts were read and relevant full papers were reviewed for data extraction. Results: From a total of 4786 full papers screened, data were extracted from 93 relevant papers. Of these, eight papers reported on long-term culture conditions (>1 month) for SSC in different livestock species, 22 papers on short-term cultures (5-15 days), 10 papers on transfection protocols, 18 papers on transplantation using different methods of preparation of livestock recipients, and five papers on donor-derived spermatogenesis. Conclusion: Optimization of SSC long-term culture systems has renewed the possibilities of utilization of these cells in gene-editing technologies to develop transgenic animals. Further, the development of genetically deficient recipients in the endogenous germline layer lends to a future possibility for the utilization of germ cell transplantation in livestock systems.

Long-term culture system for deep-sea mussels Gigantidas childressi

The simulation of deep-sea conditions in laboratories is technically challenging but necessary for experiments that aim at a deeper understanding of physiological mechanisms or host-symbiont interactions of deep-sea organisms. In a proof-of-concept study, we designed a recirculating system for long-term culture (>2 years) of deep-sea mussels Gigantidas childressi (previously Bathymodiolus childressi). Mussels were automatically (and safely) supplied with a maximum stable level of ~60 μM methane in seawater using a novel methane-air mixing system. Experimental animals also received daily doses of live microalgae. Condition indices of cultured G. childressi remained high over years, and low shell thickness growth could be detected, which is indicative of positive energy budgets. Using stable isotope data, we demonstrate that G. childressi in our culture system gained energy, both, from digestion of methane oxidizing endosymbionts and from digesting particulate food (microalgae). Limitations of the system, as well as opportunities for future experimental approaches involving deep-sea mussels are discussed.

Cellular feasibility study of Acutodesmus obliquus

The development and validation of efficient microalgae conservation methods is essential for the establishment and constitution of long-term culture collections, as well as for programming for breeding and possible genetic modification of algae. However, each species of microalgae demonstrates responses considered unpredictable, and thus, making it difficult to standardize universal methods so far. The results presented in this study indicate time as a limiting factor for the conservation of A. obliquus, in the methods of conservation by refrigeration, freezing and freezing with the use of cryoprotectants, such as DMSO, Gli and Poly. Freezing with the use of cryoprotective substances demonstrated satisfactory efficiency in the conservation of microalgae, and thus, surprisingly, in conservation by refrigeration. As already known, conservation by freezing only showed low efficiency, since the cells are broken by the formation of ice crystals in their interior, and thus, making the microalgae conservation unfeasible for a longer period. The study effectively showed the conservation of A. obliquus by refrigeration and freezing with the use of cryoprotective substances mentioned in this work.

Isolation and Expansion of Mesenchymal Stem/Stromal Cells, Functional Assays and Long-Term Culture Associated Alterations of Cellular Properties

Mesenchymal stem cell/stromal cells (MSCs) can differentiate into a variety of cell types, including osteocytes, adipocytes and chondrocytes. MSCs are present in the multiple types of adult tissue, such as bone marrow, adipose tissue, and various neonatal birth-associated tissues. Given their self-renewal and differentiation potential, immunomodulatory and paracrine properties, and lacking major histocompatibility complex (MHC) class II molecules, MSCs have attracted much attention for stem cell-based translational medicine research. Due to a very low frequency in different types of tissue, MSCs can be isolated and expanded in vitro to derive sufficient cell numbers prior to the clinical applications. In this chapter, the methodology to obtain primary bone marrow-derived MSCs as well as their in vitro culture expansion will be described. To assess the functional properties, differentiation assays, including osteogenesis, chondrogenesis and adipogenesis, 3-D culture of MSCs and co-culture of MSCs and tumor cells are also provided. Finally, the long-term culture associated alterations of MSCs, such as replicative senescence and spontaneous transformation, will be discussed for better understanding of the use of MSCs at the early stages for safe and effective cell-based therapy.

Epigenetic Drifts during Long-Term Intestinal Organoid Culture

Organoids retain the morphological and molecular patterns of their tissue of origin, are self-organizing, relatively simple to handle and accessible to genetic engineering. Thus, they represent an optimal tool for studying the mechanisms of tissue maintenance and aging. Long-term expansion under standard growth conditions, however, is accompanied by changes in the growth pattern and kinetics. As a potential explanation of these alterations, epigenetic drifts in organoid culture have been suggested. Here, we studied histone tri-methylation at lysine 4 (H3K4me3) and 27 (H3K27me3) and transcriptome profiles of intestinal organoids derived from mismatch repair (MMR)-deficient and control mice and cultured for 3 and 20 weeks and compared them with data on their tissue of origin. We found that, besides the expected changes in short-term culture, the organoids showed profound changes in their epigenomes also during the long-term culture. The most prominent were epigenetic gene activation by H3K4me3 recruitment to previously unmodified genes and by H3K27me3 loss from originally bivalent genes. We showed that a long-term culture is linked to broad transcriptional changes that indicate an ongoing maturation and metabolic adaptation process. This process was disturbed in MMR-deficient mice, resulting in endoplasmic reticulum (ER) stress and Wnt activation. Our results can be explained in terms of a mathematical model assuming that epigenetic changes during a long-term culture involve DNA demethylation that ceases if the metabolic adaptation is disturbed.