Isolatie en Verrijking van Human Adipose-afgeleide stromale cellen voor 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 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
Representative Results
Discussion
Momenteel is de isolatie van homogene subpopulaties van ASC's uit de SVF van menselijk vetweefsel blijft een uitdagende wel wenselijk doel. Isolatie van pro-osteogene ASC subpopulaties is bijzonder wenselijk, omdat dergelijke cellen kunnen worden gebruikt om de vorming en homeostase van skeletale weefsels te bestuderen. De SVF van vetweefsel herbergt opmerkelijke heterogeniteit wat celcapaciteit en differentiatie potentieel stam. 11 De moleculaire basis voor deze heterogeniteit is niet begrepen worden uit…
Declarações
The authors have nothing to disclose.
Acknowledgements
Deze studie werd ondersteund door de National Institutes of Health Research subsidie R01-DE021683-01 en de National Institutes of Health Research subsidie R01-DE019434 aan MTL; Howard Hughes Medical Institute Research Fellowship aan MTCDCW werd gesteund door de ACS Franklin Martin Faculty Research Fellowship, De Hagey Laboratory for Pediatric Regenerative Medicine, en de Stanford University Child Health Research Institute Faculteit Scholar Award.
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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