Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

William C.W. Chen; Arman Saparov; Mirko Corselli; Mihaela Crisan; Bo Zheng; Bruno P&#233;ault; Johnny Huard

doi:10.3791/51195

Aislamiento de Vaso sanguíneo derivado Multipotentes Precursores de músculo esquelético humano

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Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Aislamiento de Vaso sanguíneo derivado Multipotentes Precursores de músculo esquelético humano

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10:52 min

August 21, 2014

DOI: 10.3791/51195-v

William C.W. Chen¹, Arman Saparov^2,3, Mirko Corselli⁴, Mihaela Crisan⁵, Bo Zheng⁶, Bruno Péault^7,8, Johnny Huard⁹

¹Stem Cell Research Center, Department of Bioengineering and Orthopedic Surgery,University of Pittsburgh, ²Department of Orthopedic Surgery,University of Pittsburgh, ³Nazarbayev University Research and Innovation System,Nazarbayev University, ⁴Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital and the Orthopaedic Hospital Research Center,University of California at Los Angeles, ⁵Department of Cell Biology,Erasmus MC Stem Cell Institute, ⁶OHSU Center for Regenerative Medicine,Oregon Health & Science University, ⁷Centre for Cardiovascular Science and MRC Centre for Regenerative Medicine,Queen's Medical Research Institute and University of Edinburgh, ⁸David Geffen School of Medicine and the Orthopaedic Hospital Research Center,University of California at Los Angeles, ⁹Stem Cell Research Center, Department of Orthopedic Surgery and McGowan Institute for Regenerative Medicine,University of Pittsburgh

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Overview

This article describes a method for isolating and purifying multipotent precursor cells from human blood vessels in skeletal muscle. The procedure allows for the simultaneous purification of myogenic endothelial cells, pericytes, and adventitial cells from different structural layers of blood vessels.

Key Study Components

Area of Science

Regenerative medicine
Cell biology
Stem cell research

Background

Human skeletal muscle contains multi-lineage precursor populations.
These precursor cells have potential applications in regenerative therapies.
Isolation of these cells can enhance understanding of their properties.
Blood vessels serve as a source for these multipotent cells.

Purpose of Study

To isolate specific subsets of blood vessel-derived precursor cells.
To purify myogenic endothelial cells, pericytes, and adventitial cells.
To analyze the purity and characteristics of these cell populations.

Methods Used

Dissociation of skeletal muscle biopsy using collagenase digestion.
Labeling of single cell suspension with fluorescence-conjugated antibodies.
Fluorescence-activated cell sorting (FACS) by flow cytometry.
Post-sort analysis including flow cytometry and RT-PCR.

Main Results

Successful isolation of three distinct multipotent precursor cell populations.
High purity of sorted cell subtypes confirmed through analysis.
Method demonstrates potential for studying the origin of mesenchymal stem cells.
Provides a foundation for future regenerative applications.

Conclusions

The described method effectively isolates precursor cells from human skeletal muscle.
These cells can be utilized for further research in regenerative medicine.
Understanding these populations may lead to advancements in therapeutic strategies.

Frequently Asked Questions

What are multipotent precursor cells?

Multipotent precursor cells are cells that can differentiate into multiple cell types, particularly in the context of tissue regeneration.

How are the precursor cells isolated?

The cells are isolated using collagenase digestion followed by fluorescence-activated cell sorting (FACS).

What is the significance of these cells?

These cells have potential applications in regenerative medicine, particularly for muscle repair and regeneration.

What types of cells are purified in this study?

The study purifies myogenic endothelial cells, pericytes, and adventitial cells from blood vessels.

What techniques are used for analysis post-isolation?

Post-isolation analysis includes flow cytometry and RT-PCR to confirm cell purity and characteristics.

Can this method be applied to other tissues?

While this method is specific to skeletal muscle, similar techniques may be adapted for other tissues.

Los vasos sanguíneos dentro del puerto músculo esquelético varias poblaciones precursoras de múltiples linajes humanos que son ideales para aplicaciones regenerativas. Este método de aislamiento permite la purificación simultánea de tres poblaciones de células precursoras multipotentes, respectivamente, de tres capas estructurales de los vasos sanguíneos: las células endoteliales de miogénicos íntima, pericitos de los medios de comunicación y células adventicias de la adventicia.

El objetivo general de este procedimiento es aislar y purificar subconjuntos de células precursoras multipotentes derivadas de vasos sanguíneos humanos a partir de una sola biopsia de músculo esquelético primero, disociar la biopsia muscular y digerir con una combinación de colagenasa tipo uno, dos y cuatro. A continuación, etiquete la suspensión unicelular con anticuerpos conjugados con fluorescencia que reconocen una combinación de marcadores de superficie celular seleccionados. A continuación, realice la clasificación de células activadas por fluorescencia mediante citometría de flujo.

Vea y amplíe los subtipos de células ordenados en condiciones de cultivo específicas a densidades óptimas. En última instancia, se utiliza el análisis posterior a la clasificación de las células purificadas, así como la citometría de flujo posterior y el análisis R-T-P-C-R de los subconjuntos de células cultivadas para mostrar la pureza de estos subconjuntos homogéneos de BVSE. Sí, este método, ya sabes, puede responder realmente, ya sabes, ¿cuál es el origen de esas células madre de camello meso?

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