Biology subjects

Willerth, S.

Publications and source records attributed to Willerth, S..

4 recordsLinked to original sources

bioRxiv2021

A Xeno-free Media for the In Vitro Expansion of Human Spermatogonial Stem Cells

In vitro expansion of spermatogonial stem cells (SSCs) has been established using animal-derived fetal bovine serum (FBS) and bovine serum albumin (BSA). However, the use of animal components during cell culture introduces the risk of contaminating cells with pathogens, and leads to animal epitope expression, rendering them unsuitable for medical use. Therefore, this study set out to develop a xeno-free, fully defined media for the expansion of human SSCs. We show that the molecules Prostaglandin D2 (PGD-2) and Insulin-Like Growth Factor 1 (IGF-1) can replace FBS and BSA in cell culture media without loss of viability or expansion capability, and that Rho-Associated, Coiled-Coil Containing Protein Kinase (ROCK) inhibitor Y-27632 supplementation improves viability after cryopreservation. Long-term SSC cultures expanded in xeno-free, defined culture conditions shared identical protein expression profiles for well-known SSC markers, while gene expression analyses revealed a significant improvement in quiescent SSC and pan-germ markers. This xeno-free, defined formulation allows for standardized SSC culture free of animal pathogens.

bioengineering↗

bioRxiv2021

A NOVEL ORGANOID MODEL OF IN VITRO SPERMATOGENESIS USING HUMAN INDUCED PLURIPOTENT STEM CELLS

Infertility is thought to be caused by genetic mutations and dysfunction in the cellular niche where spermatogenesis takes place. An understanding of the specialized cellular processes which drive spermatogenesis is needed to develop treatments; however, the development of in vitro systems to study these cells has been hindered by our reliance on rarely available human testicular tissues for research. Human induced pluripotent stem cells (hiPSCs) can be used to derive human testicular-like cells, and thus provide an avenue for the development of in vitro testicular model systems. Therefore, this study set out to engineer a human testicular tissue model using hiPSCs for the first time. We demonstrate the ability of hiPSC-derived testicular cells to self-organize and mature into testicular-like tissues using organoid culture. Moreover, we show that hiPSC-derived testicular organoids promote testicular somatic cell maturation and spermatogenesis up to the post-meiotic spermatid stage. These hiPSC-derived testicular organoids have the potential to replace rarely available primary testicular tissues to further infertility research in an in vitro setting.

bioengineering↗

bioRxiv2021

Differentiation of Peritubular Myoid-Like Cells from Human Induced Pluripotent Stem Cells

Spermatogenesis is a complex process requiring intricate cellular interactions between multiple cell types to produce viable sperm. Peritubular myoid cells (PTMs) are smooth muscle cells that line the seminiferous tubules and play a critical role in sperm production by providing mechanical support and molecular signaling factors. In vitro investigation of their contribution to spermatogenesis and their dysfunction in infertility is currently limited by the rare accessibility of human testicular tissue for research. Therefore, this study set forth to generate an alternative source of PTMs using human induced pluripotent stem cells (hiPSCs) - adult cells that have been reprogrammed into a pluripotent state, making them capable of indefinite expansion and the regeneration of any cell type in the body. PTMs and Leydig cells arise from a common progenitor, so we hypothesized that PTMs could be derived by modifying an existing differentiation protocol for Leydig cell differentiation from hiPSCs. These hiPSC-derived cells, or hPTMs, were characterized and compared to hiPSC-derived Leydig cells (hLCs) and human primary Sertoli cells as a negative control. Our findings show that the substitution of the molecular patterning factor Platelet-Derived Growth Factor Subunit B (PDGF-BB) for Platelet-Derived Growth Factor Subunit A (PDGF-AA) in a molecule-based differentiation protocol for deriving Leydig-like cells, is sufficient to derive peritubular myoid-like cells. This study describes a method for generating PTM-like cells from hiPSCs. These cells will allow for ongoing understanding of the cellular interactions required for normal spermatogenesis in an in vitro setting.

bioengineering↗

bioRxiv2021

Microfluidic bioprinting for the in vitro generation of novel biomimetic human testicular tissues

Advances in cancer treatments have greatly improved pediatric cancer survival rates, leading to quality of life considerations and in particular fertility restoration. Accordingly, pre-pubertal patients have the option to cryopreserve testicular tissue for experimental restorative therapies, including in vitro spermatogenesis, wherein testicular tissue is engineered in vitro and spermatozoa are collected for in vitro fertilization (IVF). Current in vitro systems have been unable to reliably support the generation of spermatozoa from human testicular tissues, likely due to the inability for the dissociated testicular cells to recreate the native architecture of testicular tissue found in vivo. Recent advances in 3-D bioprinting can place cells into geometries at fine resolutions comparable to microarchitectures found in native tissues, and therefore hold promise as a tool for the development of a biomimetic in vitro system for human spermatogenesis. This study assessed the utility of bioprinting technology to recreate the precise architecture of testicular tissue and corresponding spermatogenesis for the first time. We printed testicular cell-laden hollow microtubules at similar resolutions to seminiferous tubules, and compared the results to testicular organoids. We show that the human testicular cells retain their viability and functionality post-printing, and illustrate an intrinsic ability to reorganize into their native cytoarchitecture. This study provides a proof of concept for the use of 3-D bioprinting technology as a tool to create biomimetic human testicular tissues.

bioengineering↗