Molecular characterization of hematopoietic stem cells after in vitro amplification on biomimetic 3D PDMS cell culture scaffolds

Lisa Marx-Blümel,Christian Marx,Jürgen Sonnemann,James F Beck,Jessica Frey,Frank Weise,Marco Groth,Jörg Hampl,Norman Rahnis,Linda Rothenburger,Zhao-Qi Wang,Emilio Cirri,Philipp Koch,Andreas Schober

doi:10.1038/s41598-021-00619-6

Abstract

Hematopoietic stem cell (HSC) transplantation is successfully applied since the late 1950s. However, its efficacy can be impaired by insufficient numbers of donor HSCs. A promising strategy to overcome this hurdle is the use of an advanced ex vivo culture system that supports the proliferation and, at the same time, maintains the pluripotency of HSCs. Therefore, we have developed artificial 3D bone marrow-like scaffolds made of polydimethylsiloxane (PDMS) that model the natural HSC niche in vitro. These 3D PDMS scaffolds in combination with an optimized HSC culture medium allow the amplification of high numbers of undifferentiated HSCs. After 14 days in vitro cell culture, we performed transcriptome and proteome analysis. Ingenuity pathway analysis indicated that the 3D PDMS cell culture scaffolds altered PI3K/AKT/mTOR pathways and activated SREBP, HIF1α and FOXO signaling, leading to metabolic adaptations, as judged by ELISA, Western blot and metabolic flux analysis. These molecular signaling pathways can promote the expansion of HSCs and are involved in the maintenance of their pluripotency. Thus, we have shown that the 3D PDMS scaffolds activate key molecular signaling pathways to amplify the numbers of undifferentiated HSCs ex vivo effectively.

Highlights

Hematopoietic stem cell (HSC) transplantation is successfully applied since the late 1950s
Recent studies show that the metabolism of fatty acids (FAs) and of cholesterol, which is regulated by PI3K/AKT/mechanistic target of rapamycin (mTOR) and connected sterol regulatory element-binding protein (SREBP) and HIF1α signaling pathways[27], is important to expand high numbers of undifferentiated HSCs ex vivo[8]
3D PDMS scaffolds offer the best conditions for ex vivo HSC expansion

Summary

Introduction

Hematopoietic stem cell (HSC) transplantation is successfully applied since the late 1950s. Ingenuity pathway analysis indicated that the 3D PDMS cell culture scaffolds altered PI3K/AKT/mTOR pathways and activated SREBP, HIF1α and FOXO signaling, leading to metabolic adaptations, as judged by ELISA, Western blot and metabolic flux analysis. These molecular signaling pathways can promote the expansion of HSCs and are involved in the maintenance of their pluripotency. Several approaches were done to improve HSC culture conditions using optimized culture media These advanced cultivation protocols include the use of hematopoietic cytokines, developmental regulators and/or chemical compounds to maintain HSC pluripotency. Recent studies show that the metabolism of fatty acids (FAs) and of cholesterol, which is regulated by PI3K/AKT/mTOR and connected sterol regulatory element-binding protein (SREBP) and HIF1α signaling pathways[27], is important to expand high numbers of undifferentiated HSCs ex vivo[8]

Methods

Results

Conclusion