Purification and differentiation of human adipose-derived stem cells by membrane filtration and membrane migration methods

Hong Reng Lin,Cheng-Hui Liu,Kadarkarai Murugan,Kadarkarai Murugan,Akon Higuchi,Akon Higuchi,Akon Higuchi,S Suresh Kumar,Giovanni Benelli,Murugan A Munusamy,Da-Chung Chen,Han-Chow Wang,Abdullah A Alarfaj,Shih-Tien Hsu,Gwo-Jang Wu,Chao-Wen Heish,Saradaprasan Muduli,Hsing-Fen Li,Nai-Chen Cheng

doi:10.1038/srep40069

Abstract

Human adipose-derived stem cells (hADSCs) are easily isolated from fat tissue without ethical concerns, but differ in purity, pluripotency, differentiation ability, and stem cell marker expression, depending on the isolation method. We isolated hADSCs from a primary fat tissue solution using: (1) conventional culture, (2) a membrane filtration method, (3) a membrane migration method where the primary cell solution was permeated through membranes, adhered hADSCs were cultured, and hADSCs migrated out from the membranes. Expression of mesenchymal stem cell markers and pluripotency genes, and osteogenic differentiation were compared for hADSCs isolated by different methods using nylon mesh filter membranes with pore sizes ranging from 11 to 80 μm. hADSCs isolated by the membrane migration method had the highest MSC surface marker expression and efficient differentiation into osteoblasts. Osteogenic differentiation ability of hADSCs and MSC surface marker expression were correlated, but osteogenic differentiation ability and pluripotent gene expression were not.

Highlights

To over 80%, which generally indicates that the culture of SVF solution on tissue culture polystyrene (TCPS) dishes leads to the “purification of hADSCs”
MACS and FACS use antibodies to purify specific target cells such as CD166, CD105, CD90, CD73, CD44, and CD29 expressing cells (CD166+, CD105+, CD90+, CD73+, CD44+, and CD29+ cells). hADSCs isolated by MACS or FACS can be used for research, but not for clinical applications, because antibodies are typically produced using animal-derived materials and make isolation of large quantity of hADSCs extremely expensive. hADSCs as well as human bone marrow stem cells are commonly used at a dose of 106–107 cells/kg in clinical applications[7,8,25,26]
Mesenchymal stem cell (MSC) isolated from human tissues, such as hADSCs, are known to possess heterogeneous characteristics, including various genotypes and differentiation abilities. hADSCs purified by different methods have different levels of stemness and purity

Summary

Introduction

To over 80%, which generally indicates that the culture of SVF solution on TCPS dishes leads to the “purification of hADSCs”. We found that expression of some pluripotent genes such as Oct3/4, Nanog, and Klf[4], which were typical genes used for the transduction of somatic cells in reprogramming into hiPSCs1–4, decreased after culture of cells in SVF solution on TCPS in our previous study[16], whereas the pluripotent gene expression of the cells in SVF solution could be maintained after purification by filtration via porous membranes[16] These surprising results indicate that hADSC characteristics depend on the method used for isolation of hADSCs from SVF solution. The migrated cells expressed MSC surface markers at higher levels than cells in SVF solution after culture on TCPS or PS dishes (cells purified by the conventional culture method)[16]. The goal of this study is to find the optimal conditions to purify hADSCs having high pluripotency and differentiation ability using the membrane filtration method and/or membrane migration method, which show better performance than hADSCs purified from SVF solution by the conventional culture method

Objectives

Methods

Results

Conclusion