Critical Analysis of cGMP Large-Scale Expansion Process in Bioreactors of Human Induced Pluripotent Stem Cells in the Framework of Quality by Design

Background The mechanisms underlying human germ cell development and infertility remain largely unknown due to bioethical issues and the shortage of experimental materials. Therefore, an effective in vitro induction system of human primordial germ-like cells (hPGCLCs) from human pluripotent stem cells (hPSC) is in high demand. The current strategies used for the generation of hPGCLCs are not only costly but also difficult to perform at a large scale, thereby posing barriers to further research. In this study, we attempted to solve these problems by providing a new 3D culture system for hPGCLC differentiation. Methods The efficiency and relative yield of a methylcellulose (MC)-based 3D hPGCLC induction system were first compared with that of a conventional U96 system. Then, we examined the gene expression of germ cell marker genes and the key epigenetic modifications of the EpCAM-/INTEGRINα6-high cells from the 3D MC induction system and the U96 system via quantitative PCR and immunofluorescence. Finally, the reliability of the MC-based 3D hPGCLC induction system was evaluated via the generation of induced pluripotent stem cells (iPSCs) from the testicular cells of one patient with obstructive azoospermia (OA) and followed by the subsequent differentiation of iPSCs into the germ cell lineage. Results In the present study, we demonstrated that the 3D MC induction system combined with low-cell attachment plates facilitated the generation of hPGCLCs at a large scale. We found that the hPGCLCs generated via the MC system shared similar characteristics to that via the U96 system in terms of the gene expression profiles, germ cell-specific markers, epigenetic modification states and cellular states. In addition, hPGCLCs from iPSCs derived from one OA patient were generated with high efficiency via the present 3D MC induction system. Discussion The in vitro induction of hPGCLCs from human embryonic stem cells (hESCs)/human induced pluripotent stem cells (hiPSCs) has significant implications in exploring the underlying mechanisms of the origin and specification of hPGCs and the epigenetic programming of the human germ line as well as treating male infertility. Here, we developed a simple and efficient 3D induction system to generate hPGCLCs from hESCs/hiPSCs at a large scale, which facilitated the study of human germ cell development and stem cell-based reproductive medicine.

Download Full-text

Macrophages Derived From Human Induced Pluripotent Stem Cells: The Diversity of Protocols, Future Prospects, and Outstanding Questions

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.640703 ◽

2021 ◽

Vol 9 ◽

Author(s):

Irina Lyadova ◽

Tatiana Gerasimova ◽

Tatiana Nenasheva

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Detailed Analysis ◽

Pluripotent Stem Cells ◽

Large Scale ◽

Therapeutic Strategies ◽

Future Prospects ◽

Current Review ◽

Induced Pluripotent ◽

Immortalized Cell

Macrophages (Mφ) derived from induced pluripotent stem cells (iMphs) represent a novel and promising model for studying human Mφ function and differentiation and developing new therapeutic strategies based on or oriented at Mφs. iMphs have several advantages over the traditionally used human Mφ models, such as immortalized cell lines and monocyte-derived Mφs. The advantages include the possibility of obtaining genetically identical and editable cells in a potentially scalable way. Various applications of iMphs are being developed, and their number is rapidly growing. However, the protocols of iMph differentiation that are currently used vary substantially, which may lead to differences in iMph differentiation trajectories and properties. Standardization of the protocols and identification of minimum required conditions that would allow obtaining iMphs in a large-scale, inexpensive, and clinically suitable mode are needed for future iMph applications. As a first step in this direction, the current review discusses the fundamental basis for the generation of human iMphs, performs a detailed analysis of the generalities and the differences between iMph differentiation protocols currently employed, and discusses the prospects of iMph applications.

Download Full-text