PLoS One. 2013 Jun 5;8(6):e64752. doi: 10.1371/journal.pone.0064752.

Awakened by cellular stress: isolation and characterization of a novel population of pluripotent stem cells derived from human adipose tissue.

Heneidi S, Simerman AA, Keller E, Singh P, Li X, Dumesic DA, Chazenbalk G.

Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington, District of Columbia, United States of America.

 

Abstract

Advances in stem cell therapy face major clinical limitations, particularly challenged by low rates of post-transplant cell survival. Hostile host factors of the engraftment microenvironment such as hypoxia, nutrition deprivation, pro-inflammatory cytokines, and reactive oxygen species can each contribute to unwanted differentiation or apoptosis. In this report, we describe the isolation and characterization of a new population of adipose tissue (AT) derived pluripotent stem cells, termed Multilineage Differentiating Stress-Enduring (Muse) Cells, which are isolated using severe cellular stress conditions, including long-term exposure to the proteolytic enzyme collagenase, serum deprivation, low temperatures and hypoxia. Under these conditions, a highly purified population of Muse-AT cells is isolated without the utilization of cell sorting methods. Muse-AT cells grow in suspension as cell spheres reminiscent of embryonic stem cell clusters. Muse-AT cells are positive for the pluripotency markers SSEA3, TR-1-60, Oct3/4, Nanog and Sox2, and can spontaneously differentiate into mesenchymal, endodermal and ectodermal cell lineages with an efficiency of 23%, 20% and 22%, respectively. When using specific differentiation media, differentiation efficiency is greatly enhanced in Muse-AT cells (82% for mesenchymal, 75% for endodermal and 78% for ectodermal). When compared to adipose stem cells (ASCs), microarray data indicate a substantial up-regulation of Sox2, Oct3/4, and Rex1. Muse-ATs also exhibit gene expression patterns associated with the down-regulation of genes involved in cell death and survival, embryonic development, DNA replication and repair, cell cycle and potential factors related to oncogenecity. Gene expression analysis indicates that Muse-ATs and ASCs are mesenchymal in origin; however, Muse-ATs also express numerous lymphocytic and hematopoietic genes, such as CCR1 and CXCL2, encoding chemokine receptors and ligands involved in stem cell homing. Being highly resistant to severe cellular stress, Muse-AT cells have the potential to make a critical impact on the field of regenerative medicine and cell-based therapy.

PMID: 23755141

 

Supplements:

Pluripotent stem cells, defined as differentiating into cells of all three embryonic germ layers and having the capacity for self-renewal, have taken center stage as the most noteworthy variety of stem cells attributed to their potential regenerative and therapeutic applications. Unfortunately, they often face a significant obstacle, which has thus precluded their clinical use. As seen in the cases of embryonic stem cells (ES) and induced pluripotent stem cells (iPS), pluripotent stem cells characteristically give rise to teratomas in vitro due to their uncontrolled pluripotency and self-renewing properties.

In 2010, Multilineage Differentiating Stress Enduring (Muse) cells were isolated from bone marrow and skin fibroblasts under severe cellular stress conditions. Unlike their ES and iPS, these stem cells negate teratoma formation but retain the self-renewing properties endowed to them by their pluripotency. When applied to an animal model, Muse cells were able to successfully generate skin, muscle and liver tissue without forming teratomas. Low telomerase activity has been identified as a key player in this pluripotent versus non-tumorigenic anomaly. It has been reported iPS cells are actually generated exclusively derived from Muse cells upon the introduction of the transcriptional genes Oct-3/4, SOX2, c-Myc, and Klf4 (the so-called “Yamanaka factors”). Gene analysis shows that classical markers of tumorigenesis are highly expressed in iPS cells derived from Muse cells as compared to naïve Muse cells. In contrast, genes involved in tumor suppression, are highly expressed in Muse cells versus iPS cells derived from Muse cells. This delicate balance may explain the degree in which the induction of the Yamanaka factors is responsible tumorigenesis and the propensity for teratoma formation inherent in iPS cells but not Muse cells, however further studies are required to elucidate this distinction.

In isolating stem cells from human adipose tissue lipoaspirate material, imposing alternate, high stress conditions, our research team at the University of California, Los Angeles, too successfully isolated Muse cells, specifically termed Muse-AT cells.  Muse-AT cells, as compared to Muse cells isolated from alternate sources throughout the body, present themselves as the most promising stem cell source for application in regenerative medicine. Practically speaking, lipoaspirate material is easily accessible, abundant, and painlessly, routinely and non-invasively extracted from the human body for both medical and cosmetic purposes. Previously, investigators have been hindered by a low yield of Muse cells from other sources, including dermal fibroblasts and bone marrow, as Muse cells make up only 1-3% of adult tissue. Hundreds of millions of adipose cells can be extracted from a mere 1-2 liters of tissue, enhancing the number of extractable Muse-AT cells.

Most uniquely, Muse cells exist in a quiescent state in the human body prior to being disrupted, or awakened as we like to think of it, by cellular stress. This has two major implications for the study of Muse cells. First, as the induction of a high-stress environment is imperative to Muse cell activation from their quiescent state, they are inherently conditioned to endure and thrive when transplanted into the harsh milieu of the recipient site in vivo. The clinical application of stem cells has often been impeded by a rate of survival, less than 3%, when exposed to the high stress engraftment environment. The intrinsic resilience of Muse cells to a high stress environment supports their ultimate translational application for tissue regeneration in vivo. Genetic analysis has revealed that Muse cells share critical genes, pertinent to the maintenance of, and mobilization from, quiescence, with cancer stem cells, making them an ideal source of information for the study of the world’s most elusive condition.

Muse cells could also shed light on anti-aging treatments. It is commonly understood that the aging of human tissue is directly related to an increase in oxidative stress. DNA mutation and degradation contribute to the increasingly harsh environment of the aging body. As Muse cells are inherently resistant to cellular stress, and genetically resilient to DNA damage, their application for the investigation of age-related and degenerative diseases is both relevant and promising.

Current studies utilize iPS as an innovative tool for drug discovery, as they model human physiology and it has been shown that various diseases can be successfully detected in differentiated adult pluripotent stem cells in vitro, providing a model for drug treatments. iPS cells have been employed for this purpose, however, as their capacity for entire genetic reprogramming remains controversial, there exists a demand which Muse cells have the potential to fulfill. Furthermore, because Muse cells are “natural pluripotent stem cells,” present in any  tissue of the body, can be assessed at every stage of differentiation, from progenitor to terminally differentiated cell, allowing the full scope of a drug’s cellular impact to be studied. As they are both pluripotent and forgo the necessity of genetic induction or manipulation, Muse cells are excellent candidates for use towards the drug discovery process.

There currently exists a single report with regards to the isolation of Muse cells in an animal model. In 2013, a Chinese research group successfully isolated Muse cells from goat-skin fibroblasts, showing their efficiency for use in somatic nuclear transfer. Confirming the existence of Muse cells in other mammalian species is a major step in the investigation of their potential and will undoubtedly inspire further animal studies to demonstrate their applicability for stem cell therapies.

Harvesting Muse cells for the purpose of autologous stem cell therapies could prove useful for the regeneration of any type of tissue present in the human body and for the treatment of an infinite list of diseases, including neurological and immune disorders, and acute injuries to critical organs such as the heart and brain. With the potential to revolutionize the fields of regenerative stem cell therapy, cancer research and drug discovery, to name a few, Muse cells have emerged as a source of boundless investigation and innovation, revitalizing the conversation surrounding the application of pluripotent stem cells in regenerative medicine.

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