This study was supported by National Institutes of Health Research grant R01-DE021683-01 and National Institutes of Health Research grant R01-DE019434 to M.T.L.; Howard Hughes Medical Institute Research Fellowship to M.T.C. D.C.W was supported by the A.C.S Franklin Martin Faculty Research Fellowship, The Hagey Laboratory for Pediatric Regenerative Medicine, and the Stanford University Child Health Research Institute Faculty Scholar Award.

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:
<ol>
	<li>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 ...

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None of the authors have a financial interest in any of the products, devices, or drugs mentioned in this manuscript. None of the authors have any competing financial interest to report.

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...

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.

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			<td style="width:112px;">15140-122</td>
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			<td height="21" style="height:21px;">PBS, pH 7.4</td>
			<td style="width:152px;">Gibco</td>
			<td>10010-023</td>
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			<td height="21" style="height:21px;">Hank&#39;s Balanced Salt Solution, 1X</td>
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			<td>21-023-CV</td>
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			<td height="21" style="height:21px;">Fetal Bovine Serum, Certified, US Origin</td>
			<td style="width:152px;">Gibco</td>
			<td>16000-044</td>
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			<td height="21" style="height:21px;">Collagenase from&#160;Clostridium histolyticum</td>
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			<td>C0130-5G</td>
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			<td style="width:152px;">Stem Cell Technologies</td>
			<td>7920</td>
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			<td height="21" style="height:21px;width:275px;">APC Mouse Anti-Human CD90</td>
			<td style="width:152px;">BD Pharmingen</td>
			<td style="width:112px;">559869</td>
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			<td height="42" style="height:42px;width:275px;">FITC Mouse anti-Human CD105 (Endoglin)</td>
			<td style="width:152px;">BD Pharmingen</td>
			<td style="width:112px;">561443</td>
			<td style="width:79px;"></td>
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			<td height="42" style="height:42px;width:275px;">Anti-Human CD45 eFluor 450 (Pacific Blue replacement)&#160;</td>
			<td style="width:152px;">eBioscience</td>
			<td style="width:112px;">48-9459-41</td>
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			<td height="21" style="height:21px;width:275px;">Anti-Human CD34 APC</td>
			<td style="width:152px;">eBioscience</td>
			<td style="width:112px;">17-0349-41</td>
			<td style="width:79px;"></td>
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			<td height="21" style="height:21px;width:275px;">Anti-Human CD31 (PECAM-1) PE</td>
			<td style="width:152px;">eBioscience</td>
			<td style="width:112px;">12-0319-41</td>
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			<td height="21" style="height:21px;width:275px;">Streptavidin PE-Cyanine7</td>
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			<td style="width:112px;">25-4317-82</td>
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			<td height="21" style="height:21px;width:275px;">BD FACS Aria II instrument</td>
			<td style="width:152px;">BD Biosciences</td>
			<td style="width:112px;"></td>
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			<td height="21" style="height:21px;width:275px;">BD FACSDiva Software</td>
			<td style="width:152px;">BD Biosciences</td>
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isolation and enrichment of human adipose-derived stromal cells for enhanced osteogenesis

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.

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...

Watch this Scientific Journal Video about Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis at JoVE.com

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

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.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

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11.3K Görüntüleme.  Stanford University School of Medicine. 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.