Using Advanced Cell Culture Techniques to Differentiate Pluripotent Stem Cells and Recreate Tissue Structures Representative of Teratoma Xenografts.

E. Knight,L. A. Smith,D. D. G. Owens,R. H. Quelch,A. Hidalgo Aguilar,S. A. Przyborski

doi:10.3389/fcell.2021.667246

Abstract

Various methods are currently used to investigate human tissue differentiation, including human embryo culture and studies utilising pluripotent stem cells (PSCs) such as in vitro embryoid body formation and in vivo teratoma assays. Each method has its own distinct advantages, yet many are limited due to being unable to achieve the complexity and maturity of tissue structures observed in the developed human. The teratoma xenograft assay allows maturation of more complex tissue derivatives, but this method has ethical issues surrounding animal usage and significant protocol variation. In this study, we have combined three-dimensional (3D) in vitro cell technologies including the common technique of embryoid body (EB) formation with a novel porous scaffold membrane, in order to prolong cell viability and extend the differentiation of PSC derived EBs. This approach enables the formation of more complex morphologically identifiable 3D tissue structures representative of all three primary germ layers. Preliminary in vitro work with the human embryonal carcinoma line TERA2.SP12 demonstrated improved EB viability and enhanced tissue structure formation, comparable to teratocarcinoma xenografts derived in vivo from the same cell line. This is thought to be due to reduced diffusion distances as the shape of the spherical EB transforms and flattens, allowing for improved nutritional/oxygen support to the developing structures over extended periods. Further work with EBs derived from murine embryonic stem cells demonstrated that the formation of a wide range of complex, recognisable tissue structures could be achieved within 2–3 weeks of culture. Rudimentary tissue structures from all three germ layers were present, including epidermal, cartilage and epithelial tissues, again, strongly resembling tissue structure of teratoma xenografts of the same cell line. Proof of concept work with EBs derived from the human embryonic stem cell line H9 also showed the ability to form complex tissue structures within this system. This novel yet simple model offers a controllable, reproducible method to achieve complex tissue formation in vitro. It has the potential to be used to study human developmental processes, as well as offering an animal free alternative method to the teratoma assay to assess the developmental potential of novel stem cell lines.

Highlights

The study of human tissue differentiation continues to be of significant interest and importance to a wide range of biological fields, including human developmental biology, stem cell science, and regenerative medicine
The tissue structures formed using this new approach were comparable to xenograft teratoma tissues derived from the same cell line in vivo. We demonstrate that this novel in vitro system provides a controllable and robust method to study and direct the formation of rudimentary human tissues over time, as well as providing a potential animal free alternative to the traditional teratoma assay for the assessment of stem cell pluripotency
While the overall arrangement of tissue structures is disorganised within the xenograft (A), specific tissue types are clearly identifiable with distinct cellular morphologies and organisation, and their identities may be further validated using histological stains

Summary

Introduction

The study of human tissue differentiation continues to be of significant interest and importance to a wide range of biological fields, including human developmental biology, stem cell science, and regenerative medicine. Notable progress is being made, many events and mechanisms during this process remain to be clarified, with a wide range of techniques used to investigate these processes. Both in vivo and in vitro approaches are employed; each has its own unique advantages and limitations regarding the method and the information that can be elucidated (see Table 1 for brief overview and comparisons). General developmental processes permit the transposition of data obtained from animal studies to human differentiation events, for example, the identification of general mechanisms, such as the formation of the body axis. There is a commitment to decrease the use of animal based models in favour of reducing, refining and replacing these experiments with appropriate and sustainable alternatives (Festing and Wilkinson, 2007)

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