Name: isolation and enrichment of human adipose-derived stromal cells for enhanced osteogenesis
Uploaded: 2015-01-12T14:30:00
Description: Watch this Scientific Journal Video about Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis at JoVE.com

Este estudo foi financiado pelo National Institutes of Health Research concessão R01-DE021683-01 e National Institutes of Health Research concessão R01-DE019434 para MTL; Howard Hughes Medical Institute Research Fellowship para MTCDCW foi apoiado pela ACS Franklin Martin Faculdade Research Fellowship, O Laboratório Hagey for Pediatric Regenerative Medicine, e do Instituto de Pesquisa de Saúde Infantil da Universidade de Stanford Faculdade Scholar Award.

NOTA: Todas as amostras dos pacientes foram obtidos com o consentimento informado, e os protocolos experimentais foram revistos e aprovados pela Stanford University Institutional Review Board (Protocolo nº 2188 e # 9999). 1. Isolamento celular e Cultura: <ol><li> Obter tecido adiposo subcutâneo humano de pacientes saudáveis ​​do sexo feminino submetidos a lipoaspiração eletiva do abdômen, flanco, e / ou região de coxa sob anestesia local / geral. Certifique-se de que Institution...

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Actualmente, o isolamento de subpopulações homogéneas de ASC de SVF de tecido adiposo humano continua a ser um desafio embora objectivo desejável. Isolamento de subpopulações ASC pró-osteogénicas é particularmente desejável, uma vez que tais células podem ser utilizadas para estudar a formação e a homeostase do tecido esquelético. No entanto, o SVF de tecido adiposo abriga heterogeneidade significativa no que diz respeito a conter capacidade celular e potencial de diferenciação. 11 A base mole...

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:152px;">BD Pharmingen</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>
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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>
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			<td style="width:152px;">BD Biosciences</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.

Usando CD90 como um marcador para células com melhores resultados de osteogénese no isolamento de uma população altamente enriquecidas de ASC humanos (Figura 1A, 1B). ASC foram coradas com azul-Pacífico CD45 conjugado anti-humano, conjugado com FITC anti-CD105 humano, e CD90 anti-humano conjugado com APC. Após a separação, o nível de pureza foi superior a 98%, tal como quantificado por análise post-tipo. Definição de grupos de células com base em perfis de trans...

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.

Isolamento e Enriquecimento da Human adiposo-derivadas células estromais para Osteogenesis Aprimorado

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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