Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

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 s....

Protocol

NOTE: All patient samples were obtained with informed consent, and experimental protocols were reviewed and approved by Stanford University Institutional Review Board (Protocol #2188 and #9999).

1. Cell Isolation and Culture:

1. Obtain human subcutaneous adipose tissue from healthy female patients undergoing elective lipoaspiration of the abdomen, flank, and/or thigh region under local/general anesthesia. Ensure that Institutional Review Board (IRB) approval has been obtained for the protocol of isolating ASCs from human tissues, and follow institutional safety precautions while working with such materials.
2. To obtain the ....

Results

Using CD90 as a marker for cells with enhanced osteogenesis results in isolation of a highly-enriched populations of human ASCs (Figure 1A, 1B). ASCs were stained with Pacific Blue-conjugated anti-human CD45, FITC-conjugated anti-human CD105, and APC-conjugated anti-human CD90. After sorting, the level of purity was greater than 98%, as quantified by post-sort analysis.

Defining groups of cells based on transcriptional profiles allowed for prospective isolation of two novel su.......

Discussion

Currently, the isolation of homogenous subpopulations of ASCs from the SVF of human adipose tissue remains a challenging though desirable goal. Isolation of pro-osteogenic ASC subpopulations is particularly desirable, as such cells can be used to study the formation and homeostasis of skeletal tissues. However, the SVF of adipose tissue harbors significant heterogeneity with regard to stem cell capacity and differentiation potential.¹¹ The molecular basis for this heterogeneity cannot be understood from pooled.......

Materials

List of materials used in this article

Name	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-Cy7	eBioscience	25-4317-82
BD FACS Aria II instrument	BD Biosciences
BD FACSDiva Software	BD Biosciences