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

ABSTRACT: Infertility affects approximately 15% of couples, and in couples where a male factor is present 15% of these males lack production of any sperm. In order to derive novel regenerative therapies, we must first have an improved understanding of the cell-type-specific function in normal and infertile human testes that regulate spermatogenesis must be achieved. This understanding requires the ability to isolate and functionally characterize each human testis cell type in isolation and in combination within the spermatogenic niche. Similar to other human organ systems, lack of availability to obtain biopsies with viable cells to subsequently, isolate and propagate specific cell types, has stifled human spermatogenesis research. Previous researchers have utilized engineering human induced pluripotent stem cells (hiPSCs) to generate various organ specific cell types for use as in vitro models. In the human testis, previous groups have derived Leydig cells, Sertoli cells, spermatogonial stem cells (SSCs), macrophages and endothelial cells via this method. However, peritubular myoid cells (PTMs) which serve critical roles in human testis cytoarchitecture and spermatogenic regulation, remain to be derived. Therefore, this study set forth to generate hiPSC-derived PTMs to serve as a tool to model and study human PTM function and in vitro spermatogenic modelling by deriving the final missing somatic cell type ofin the human testis. PTMs and Leydig cells arise from a common progenitor, so it was 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 primary PTMs. Results show that the substitution of the molecular patterning factor Platelet-Derived Growth Factor Subunit B (PDGFBB) for Platelet-Derived Growth Factor Subunit A (PDGFAA) in a molecule-based differentiation protocol for deriving Leydig-like cells is sufficient to derive cells with peritubular myoid-like transcriptomes and smooth muscle protein expression in an hiPSC line primed for a mesoderm lineage. Results also indicate that maturation of differentiated PTMs between hiPSC lines is variable. Comparison of lineage affiliated gene expression suggests that hiPSC lines with gene expression reflecting a primed state for mesoderm lineage can more efficiently and consistently derive hPTM-like cells. Overall, these hPTMs will allow for in vitro study of their phenotypes and functional role in spermatogenesis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE219132 | GEO | 2023/02/02

REPOSITORIES: GEO

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Project description:Thiele2013 - Testis Leydig cells The model of testis Leydig cells metabolism is derived from the community-driven global reconstruction of human metabolism (version 2.02, MODEL1109130000 ). This model is described in the article: A community-driven global reconstruction of human metabolism. Thiele I, et al . Nature Biotechnology Abstract: Multiple models of human metabolism have been reconstructed, but each represents only a subset of our knowledge. Here we describe Recon 2, a community-driven, consensus 'metabolic reconstruction', which is the most comprehensive representation of human metabolism that is applicable to computational modeling. Compared with its predecessors, the reconstruction has improved topological and functional features, including ~2x more reactions and ~1.7x more unique metabolites. Using Recon 2 we predicted changes in metabolite biomarkers for 49 inborn errors of metabolism with 77% accuracy when compared to experimental data. Mapping metabolomic data and drug information onto Recon 2 demonstrates its potential for integrating and analyzing diverse data types. Using protein expression data, we automatically generated a compendium of 65 cell type-specific models, providing a basis for manual curation or investigation of cell-specific metabolic properties. Recon 2 will facilitate many future biomedical studies and is freely available at http://humanmetabolism.org/. This model is hosted on BioModels Database and identified by: MODEL1310110008 . To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models . To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Project description:Although reported more than once, the generation of pluripotent stem cell populations from human testis remains questionable. We recently showed that the so - called human adult germline stem cells (haGSCs), which were claimed to be pluripotent, showed a gene expression profile comparable to human fibroblasts, suggesting that these cells are originated from a fibroblastic cells. We could derive human testicular fibroblast cells (hTFCs) from several routine testicular biopsies, naming them human testicular fibroblastic cells (hTFCs). The true conversion of unipotent mouse GSCs, also known as spermatogonial stem cells into pluripotent stem cells was recently shown. However, this conversion was not observed for human cells so far. We suggest that hTFCs isolated from routine biopsies can help in the understanding, modeling and also regenerative medicine for various diseases after reprogramming them to human induced pluripotent stem cells (iPS). We successfully reprogrammed hTFCs into human iPS cells by transducing them retrovirally with reprogramming factors as already demonstrated for human dermal fibroblasts. These hTFCs derived iPS cells are comparable with human embryonic stem cells from global gene expression, promoter methylation, and they show pluripotency judged by in vitro and in vivo differentiation. For transcriptome profiling, 400 ng of total DNA-free RNA was used as input for labelled cRNA synthesis (Illumina TotalPrep RNA Amplification Kit - Ambion) following the manufacturer's instructions (IVT: 12h). Quality-checked cRNA samples were hybridized as biological or technical duplicates for 18 h onto human-8 v3 expression BeadChips (Illumina), washed, stained, and scanned following guidelines and using materials/instrumentation supplied/suggested by the manufacturer. 8 samples were analyzed: NCL: NCL3 hESCs grown on feeders, bulk harvest at semi-sub confluence (1 replicate) HuES6: HuES6 hESCs grown on feeders, bulk harvest at semi-sub confluence (1 replicate) HT3: Human fibroblast-like testis cells, line 3 (1 replicate) HT16: Human fibroblast-like testis cells, line 16 (1 replicate) FS: FS human foreskin fibroblasts (1 replicate) BJ: BJ human fibroblasts (1 replicate) iPS3: hiPSC line 2 derived from HT3 (1 replicate) iPS16: hiPSC line 2 derived from HT16 (1 replicate)

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

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