Unravelling Heterogeneity of Amplified Human Amniotic Fluid Stem Cells Sub-Populations.

Francesca Casciaro,Andrea Zattoni,Francesco Alviano,Silvia Zia,Mattia Forcato,Tullia Maraldi,Manuela Zavatti,Emma Bertucci,Francesca Beretti,Marco Demaria,Pierluigi Reschiglian,Barbara Roda,Matilde Yung Follo,Laura Bonsi

doi:10.3390/cells10010158

Abstract

Human amniotic fluid stem cells (hAFSCs) are broadly multipotent immature progenitor cells with high self-renewal and no tumorigenic properties. These cells, even amplified, present very variable morphology, density, intracellular composition and stemness potential, and this heterogeneity can hinder their characterization and potential use in regenerative medicine. Celector® (Stem Sel ltd.) is a new technology that exploits the Non-Equilibrium Earth Gravity Assisted Field Flow Fractionation principles to characterize and label-free sort stem cells based on their solely physical characteristics without any manipulation. Viable cells are collected and used for further studies or direct applications. In order to understand the intrapopulation heterogeneity, various fractions of hAFSCs were isolated using the Celector® profile and live imaging feature. The gene expression profile of each fraction was analysed using whole-transcriptome sequencing (RNAseq). Gene Set Enrichment Analysis identified significant differential expression in pathways related to Stemness, DNA repair, E2F targets, G2M checkpoint, hypoxia, EM transition, mTORC1 signalling, Unfold Protein Response and p53 signalling. These differences were validated by RT-PCR, immunofluorescence and differentiation assays. Interestingly, the different fractions showed distinct and unique stemness properties. These results suggest the existence of deep intra-population differences that can influence the stemness profile of hAFSCs. This study represents a proof-of-concept of the importance of selecting certain cellular fractions with the highest potential to use in regenerative medicine.

Highlights

Mesenchymal stem cells (MSCs) have the ability to support other cell types during tissue regeneration [1], to secrete various growth factors and cytokines with pro-regenerative properties [2] and to modulate immune responses [3]
Sorting of Human amniotic fluid stem cells (hAFSCs) by Celector® Technology c-kit+ -hAFSCs at passage 5–6 in culture from 10 patients were sorted using the
Since we have shown that improved proliferative potential and reduced cellular aging is associated with enhanced stemness properties in hAFSCs, we evaluated the expression of the pluripotency markers Oct4 and Nanog

Summary

Introduction

Mesenchymal stem cells (MSCs) have the ability to support other cell types during tissue regeneration [1], to secrete various growth factors and cytokines with pro-regenerative properties [2] and to modulate immune responses [3]. For this reason, MSCs therapy represents a potential intervention for a variety of dysfunctions and disorders. MSCs are present in both adult and foetal tissues, and more attention has been recently given to placental and amniotic fluid stem cells. These cells express multiple pluripotent markers [11] and differentiate into mesodermal and non-mesodermal lineages under appropriate differentiation conditions, but unlike embryonic stem cells or induced pluripotent stem cells, they do not form tumours in vivo and do not form chimeras when injected into blastocysts [12]

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