Summary

強化された骨形成のためのヒト脂肪由来間質細胞の単離および濃縮

Published: January 12, 2015
doi:

Summary

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

Abstract

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

Introduction

The heterogeneous nature of stem cell populations is not yet fully understood and remains a major impediment to the development of clinically effective stem cell-based therapeutic applications. One of the most common ways to characterize a heterogeneous population of stem cells is to employ a cell sorting method, such as fluorescence-activated cell sorting (FACS), to separate cells based on their surface marker expression profiles. As sorting methods become more complex, it becomes possible to identify more distinct functional subpopulations of cells. Microfluidic-based technologies are becoming more and more frequently utilized in analysis of gene expression at the single cell level. Multiplexed quantitative polymerase chain reaction (qPCR) within a microfluidic chip allows for effective and reliable high-resolution, single cell transcriptional analysis.1-5

In a previous study using single cell transcriptional profiling of 48 genes, considerable transcriptional heterogeneity was observed among ASCs.6 However, the distribution of genes MSX2, BMP-5, BMP-7, ALP, OCN, RUNX2 exhibited a strong association with a cluster of cells possessing highly osteogenic transcriptional profiles. To isolate cells according to this osteogenic gene expression profile, surface antigen expression patterns were correlated with transcription patterns and surface marker expression of endoglin (CD105) was subsequently discovered to closely correlate with enhanced osteogenic differentiation potential of ASCs. Independent of CD105 expression, expression of surface receptor Thy-1 (CD90), a glycosyl-phosphatidylinositol-linked membrane protein previously shown by Chen et al. to be associated with osteoprogenitor cells, was also correlated with osteogenic gene expression.6,7 These findings provide the opportunity to prospectively isolate subpopulations within the larger heterogeneous pool of ASCs with increased osteogenic capacity for cell-based bone tissue engineering applications.

Protocol

注:すべての患者の試料はインフォームドコンセントを得て、実験的なプロトコルは、スタンフォード大学の治験審査委員会(プロトコル#2188と#9999)によって審査され、承認された。 1.細胞の単離および培養: 一般/局所麻酔下で腹部、脇腹、および/または大腿部領域の選択科目lipoaspirationを受けた健康女性患者からのヒト皮下脂肪組織を入手します。治験審…

Representative Results

ヒトASC( 図1A、1B)の高濃縮集団の単離に強化された骨形成の結果と細胞のマーカーとしてCD90を使用した。 ASCははパシフィックブルー結合抗ヒトCD45、FITC結合抗ヒトCD105、およびAPC結合抗ヒトCD90で染色した。ポストソート分析によって定量しソートした後、純度のレベルは、98%以上であった。 2つの新規な亜集団の将来の単離のために許可された転写プロ?…

Discussion

現在、ヒト脂肪組織のSVFからASCの均質な亜集団の単離は、望ましい目標ものの依然として厳しい。このような細胞は、骨格組織の形成および恒常性を研究するために使用することができるようにプロ骨形成ASC亜集団の単離は、特に望ましい。しかし、脂肪組織のSVFは、セル容量と分化能を食い止めるに関して有意な異質性を保有する。11この異質性の分子基盤は、細胞のプールされた…

Disclosures

The authors have nothing to disclose.

Acknowledgements

この研究は、MTLに健康研究助成R01-DE021683-01と健康研究助成金R01-DE019434の国立衛生研究所の国立研究所によってサポートされていました。 MTCDCWにハワード·ヒューズ医学研究所研究員は、ACSフランクリン·マーティン学部研究フェローシップ、小児再生医療のためのHagey研究所、スタンフォード大学小児保健研究所学部奨学生賞によってサポートされていました。

Materials

Name of Reagent/Material Company Catalog Number Comments
Disposable 250 mL Conical Tubes Corning (Thomas Scientific) 2602A43
Penicillin-Streptomycin (10,000 U/mL) Gibco 15140-122
DMEM, high glucose, GlutaMAX Supplement Gibco 10566-016
PBS, pH 7.4 Gibco 10010-023
Betadine – Antiseptic Povidone/Iodine Solution Purdue  PFC-67618015017
Hank's Balanced Salt Solution, 1X Cellgro 21-023-CV
Fetal Bovine Serum, Certified, US Origin Gibco 16000-044
Collagenase from Clostridium histolyticum Sigma-Aldrich C0130-5G
ACCUTASE Cell Detachment Solution Stem Cell Technologies 7920
APC Mouse Anti-Human CD90 BD Pharmingen 559869
FITC Mouse anti-Human CD105 (Endoglin) BD Pharmingen 561443
Anti-Human CD45 eFluor 450 (Pacific Blue replacement)  eBioscience 48-9459-41
Anti-Human CD34 APC eBioscience 17-0349-41
Anti-Human CD31 (PECAM-1) PE eBioscience 12-0319-41
Streptavidin PE-Cyanine7 eBioscience 25-4317-82
BD FACS Aria II instrument BD Biosciences
BD FACSDiva Software BD Biosciences

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Cite This Article
Zielins, E. R., Tevlin, R., Hu, M. S., Chung, M. T., McArdle, A., Paik, K. J., Atashroo, D., Duldulao, C. R., Luan, A., Senarath-Yapa, K., Walmsley, G. G., Wearda, T., Longaker, M. T., Wan, D. C. Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis. J. Vis. Exp. (95), e52181, doi:10.3791/52181 (2015).

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