Awakened by Cellular Stress：Isolation and Characterization of a Novel Population of Pluripotent Stem Cells Derived from Human Adipose Tissue（1）
Saleh Heneidi, Ariel A. Simerman, Erica Keller, Prapti Singh, Xinmin Li, Daniel A. Dumesic, Gregorio Chazenbalk
Published：June 05, 2013
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
, 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.
Citation: Heneidi S, Simerman AA, Keller E, Singh P, Li X, et al. (2013) Awakened by Cellular Stress: Isolation and Characterization of a Novel Population of Pluripotent Stem Cells Derived from Human Adipose Tissue. PLoS ONE 8(6): e64752. doi:10.1371/journal.pone.0064752
Editor: Alexander V. Ljubimov, Cedars-Sinai Medical Center, United States of America
Received: February 7, 2013; Accepted: April 17, 2013; Published: June 5, 2013
Copyright: © 2013 Heneidi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Part of these studies were supported by the Department of Obstetrics/Gynecology at University of California Los Angeles and by the Eunice Kennedy Shriver National Institute of Child Health & Human Development, National Institutes of Health through cooperative agreement U54 HD071836. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Cellular stress is induced by abrupt disruption of the physiological niche: the optimal home most conducive to cell survival. Although adult stem cells have been considered an attractive source for cell therapy, their effectiveness and efficiency is hindered by a frequently low survival rate due to their exposure to a high cellular stress environment upon transplantation. This key limitation is observed when utilizing adult stem cells for regenerative purposes, as typical cell engraftment yields are extremely low (<3%). Multiple factors contribute to this low rate of cell survival, including the harsh environment of the recipient site, harboring pro-apoptotic factors including hypoxia, malnutrition, pro-inflammatory cytokines and reactive oxygen and nitrogen species. The severity of cellular stress is heightened when stem cells are administered to an acutely injured area, such as a myocardial infarction, stroke, or a peripheral ischemic injury
, as are the chances of unwanted activation or differentiation of surviving cells. It is extremely difficult to alter the environment of the damaged tissue, which necessitates a viable alternative: to improve post-transplant stem cell survival rates through the administration of a stem cell population with the adaptations necessary for survival in the hostile host environment.
One potential solution to this problem is to gradually adapt stem cells to cellular stress prior to cell delivery. It has been shown that introducing stem cells to hypoxic conditions in vitro for a duration of 24–48 hours
, also known as Hypoxia preconditioning
), provides the opportunity for these cells to adapt to low oxygen concentrations, thus increasing chances for survival upon reintroduction to hypoxic conditions in vivo. HPC is a promising solution to the severe apoptosis that accompanies transplantation as it induces an adaptive mechanism that increases the likelihood of cell survival in a pro-apoptotic microenvironment in vivo. Adult human Mesenchymal stem cells
) and adult Hematopoietic stem cells
) have similarly been shown to increase expansion, survival, and self-renewal under hypoxia conditions while maintaining the capability for multi-lineage differentiation
Another potential solution to the problem of successful delivery of stem cells to a hostile host environment is to utilize a purified population of stem cells, isolated during exposure to severe cellular stress conditions (e.g. long time incubation to proteolytic enzymes, hypoxic conditions, serum deprivation, low temperatures), for engraftment. Recently, a new stem cell population has been isolated from mesenchymal tissues such as human skin fibroblasts and bone marrow stromal cells under cellular stress conditions. These cells, termed Multilineage Differentiating Stress-Enduring (Muse
, are of Mesenchymal stem cell origin
and comprise 1–3% of the entire cell population
. Muse cells exhibit characteristics of both Mesenchymal and Pluripotent stem cells
. They are double positive for CD105
, a mesenchymal stem cell marker, and Stage specific embryonic antigen-3
), well known for the characterization of undifferentiated human embryonic stem cells (ES) from bone marrow aspirates or from cultured mesenchymal cells such as bone marrow stromal cells and dermal fibroblasts. They express Pluripotency markers including Oct3/4, Nanog and Sox2
, differentiate into cells of ectodermal, endodermal, and mesodermal lineages both in vitro and in vivo
, and have the ability to self-renew
. Advantageously, Muse cells do not appear to undergo tumorigenic proliferation
, and therefore would not be prone to produce teratomas in vivo
, nor do they induce immuno-rejection in the host upon autologous transplantation
. In addition, Muse cells are shown to home into the damage site in vivo
and spontaneously differentiate into Tissue specific cells according to the Microenvironment to contribute to Tissue regeneration when infused into the blood stream
. Therefore, they exhibit the potential to make critical contributions to tissue regeneration in the absence of restrictions attributed to the difficult extraction of bone marrow stromal cells and human skin fibroblasts, and time-consuming purification methods such as cell sorting. In order to increase the viability of Muse cells as a source of tissue regeneration, a more accessible supply must be utilized.
Harvesting human adipose tissue by Lipoaspiration is a safe and non-invasive procedure, and hundreds of millions of cells can be isolated from 1–2 liters of lipoaspirate material
. Therefore, adipose tissue could prove the ideal source for Muse cell isolation as opposed to bone marrow or dermis. Using lipoaspirate material, we developed a novel methodology for the isolation of a population of human Muse cells under Severe cellular stress conditions
(long term incubation with Proteolytic enzyme, 4°C, serum deprivation, and Hypoxia
). Purification of human Muse cells derived from adipose tissue (Muse-ATs) does not require the use of cell sorting, magnetic beads or special devices. Muse-ATs can grow either in suspension, forming cell spheres, or as adherent cells forming cell aggregates
similar to human ES cell-derived embryoid bodies as previously reported. Furthermore, Muse-AT cells express Pluripotent stem cell markers and a variety of markers indicative of all three germlines
. Upon the introduction to specific culture conditions, Muse-AT cells can differentiate to mesenchymal (adipocytes, skeletal and smooth muscle cells), endodermal (hepatocytes and biliary ducts) and ectodermal (neural cells) cell lineages both spontaneously and by differentiation induction. Immunocytochemistry and microarray data demonstrate Up-regulation of the Pluripotent stem cell markers Sox2, Oct3/4, and Rex1 in Muse-AT cells
, as compared to previously studied multipotent adipose stem cells (ASCs). Microarray analysis reveals that Muse-AT cells highly express genes involved in Cellular protection against Oxidative stress
. Additionally, these cells also exhibit up regulation of CXCL2 gene expression, a critical Chemokine involved in stem cell Homing
. Muse-AT cells display down regulation of genes involved in cell death and survival, embryonic development, organism survival, cellular assembly and organization, mitosis, DNA replication, recombination and repair
. Because lipoaspiration is a safe and non-invasive procedure and Muse-AT cell isolation requires a simple yet highly efficient purification technique, Muse-AT cells could provide an ideal source of pluripotent-like stem cells with the potential to have a critical impact on regenerative medicine and cell-based therapy.