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Human male reproductive development has a prolonged prepubertal period characterized by juvenile quiescence of germ cells with immature spermatogonial stem cell (SSC) precursors (gonocytes) present in the testis for an extended period of time. The metabolism of gonocytes is not defined. We demonstrate with mitochondrial ultrastructure studies via TEM and IHC and metabolic flux studies with UHPLC-MS that a distinct metabolic transition occurs during the maturation to SSCs. The mitochondrial ultrastructure of prepubertal human spermatogonia is shared with prepubertal pig spermatogonia. The metabolism of early prepubertal porcine spermatogonia (gonocytes) is characterized by the reliance on OXPHOS fuelled by oxidative decarboxylation of pyruvate. Interestingly, at the same time, a high amount of the consumed pyruvate is also reduced and excreted as lactate. With maturation, prepubertal spermatogonia show a metabolic shift with decreased OXHPOS and upregulation of the anaerobic metabolism-associated uncoupling protein 2 (UCP2). This shift is accompanied with stem cell specific promyelocytic leukemia zinc finger protein (PLZF) protein expression and glial cell-derived neurotropic factor (GDNF) pathway activation. Our results demonstrate that gonocytes differently from mature spermatogonia exhibit unique metabolic demands that must be attained to enable their maintenance and growth in vitro., (© 2021 Federation of American Societies for Experimental Biology.)

Normal developmental progression relies on close interactions between the embryonic and extraembryonic lineages in the pre- and peri-gastrulation stage conceptus. For example, mouse epiblast-derived FGF and NODAL signals are required to maintain a stem-like state in trophoblast cells of the extraembryonic ectoderm, while visceral endoderm signals are pivotal to pattern the anterior region of the epiblast. These developmental stages also coincide with the specification of the first heart precursors. Here, we established a robust differentiation protocol of mouse embryonic stem cells (ESCs) into cardiomyocyte-containing embryoid bodies that we used to test the impact of trophoblast on this key developmental process. Using trophoblast stem cells (TSCs) to produce trophoblast-conditioned medium (TCM), we show that TCM profoundly slows down the cardiomyocyte differentiation dynamics and specifically delays the emergence of cardiac mesoderm progenitors. TCM also strongly promotes the retention of pluripotency transcription factors, thereby sustaining the stem cell state of ESCs. By applying TCM from various mutant TSCs, we further show that those mutations that cause a trophoblast-mediated effect on early heart development in vivo alter the normal cardiomyocyte differentiation trajectory. Our approaches provide a meaningful deconstruction of the intricate crosstalk between the embryonic and the extraembryonic compartments. They demonstrate that trophoblast helps prolong a pluripotent state in embryonic cells and delays early differentiative processes, likely through production of leukemia inhibitory factor (LIF). These data expand our knowledge of the multifaceted signaling interactions among distinct compartments of the early conceptus that ensure normal embryogenesis, insights that will be of significance for the field of synthetic embryo research. [ABSTRACT FROM AUTHOR]

"To consult the statistician after an experiment is finished is often merely to ask him to conduct a postmortem examination. He can perhaps say what the experiment died of." - R.A. Fisher While this idea is relevant across research scales, its importance becomes critical when dealing with the inherently large, complex and expensive process of preparing material for cell-based therapies (CBTs). Effective and economically viable CBTs will depend on the establishment of optimized protocols for the production of the necessary cell types. Our ability to do this will depend in turn on the capacity to efficiently search through a multi-dimensional problem space of possible protocols in a timely and cost-effective manner. In this review we discuss approaches to and illustrate examples of the application of statistical design of experiments to stem cell bioprocess optimization. [ABSTRACT FROM AUTHOR]

Abstract: Human pluripotent cells such as human embryonic stem cells (hESC) are a great potential source of cells for cell-based therapies; however, directing their differentiation into the desired cell types with high purity remains a challenge. The stem cell microenvironment plays a vital role in directing hESC fate and we have previously shown that manipulation of colony size in a serum- and cytokine-free environment controls self-renewal and differentiation toward the extraembryonic endoderm lineage. Here we show that, in the presence of bone morphogenetic protein 2 and activin A, control of colony size using a microcontact printing technology is able to direct hESC fate to either the mesoderm or the endoderm lineage. Large, 1200-μm-diameter colonies give rise to mesoderm, while small 200-μm colonies give rise to definitive endoderm. This study links, for the first time, cellular organization to pluripotent cell differentiation along the mesoderm and endoderm lineages. [Copyright &y& Elsevier]

Male survivors of childhood cancer are at risk of suffering from infertility in adulthood because of gonadotoxic chemotherapies. For adult men, sperm collection and preservation are routine procedures prior to treatment; however, this is not an option for pre-pubertal children. From young boys, a small biopsy may be taken before chemotherapy, and spermatogonia may be propagated in vitro for future transplantation to restore fertility. A robust system that allows for scalable expansion of spermatogonia within a controlled environment is therefore required. Stirred suspension culture has been applied to different types of stem cells but has so far not been explored for spermatogonia. Here, we report that pre-pubertal porcine spermatogonia proliferate more in bioreactor suspension culture, compared with static culture. Interestingly, oxygen tension provides an avenue to modulate spermatogonia status, with culture under 10% oxygen retaining a more undifferentiated state and reducing proliferation in comparison with the conventional approach of culturing under ambient oxygen levels. Spermatogonia grown in bioreactors upregulate the Wnt/ β-catenin pathway, which, along with enhanced gas and nutrient exchange observed in bioreactor culture, may synergistically account for higher spermatogonia proliferation. Therefore, stirred suspension bioreactors provide novel platforms to culture spermatogonia in a scalable manner and with minimal handling. [ABSTRACT FROM AUTHOR]