dult stem cells are rare cells scattered throughout our bodies that, when called into action, have the ability to proliferate and give rise to progeny that can differentiate into a number of different cell types. Mesenchymal stem cells (MSCs) are one type of adult stem cell that can be isolated from umbilical cord blood. After their isolation, MSCs must be extensively expanded in cell cultures in order to generate numbers that are sufficient for clinical use. Unfortunately, by the time sufficient numbers of MSCs have been generated, there is a danger that the cell population may contain a significant number of cells that are no longer able to proliferate (a state of cellular senescence), as well as some genomically unstable cells that have somehow managed to bypass senescence. Collaborative studies being carried out in our laboratories at the NJ Institute for Successful Aging in Stratford and the Coriell Institute in Camden have focused on elucidating these potentially deleterious changes, with the aim of developing useful strategies that will document the safety of these cells for therapeutic use.
Humans are large bodied animals with a long life span. We start our lives as a single cell, but when we achieve our adult size and maximal tissue complexity, our bodies are composed of roughly 16 trillion cells that fall into approximately 500 different categories of cell types. This is a remarkable feat by any standard; especially considering that the entire human genome that encodes everything we have been, are, and will be must be completely and accurately duplicated in our cells with each mitotic division. However, the mitotic demand on our cells does not stop when we achieve full size at adulthood. Because the life span of our body is much longer than that of most of its individual constituent cells, an efficient system of cell renewal and replacement has evolved. Examples of this are found on the surfaces of our skin and intestinal lining, where older cells are constantly being shed and replaced by new cells. Individual cells in other organs, such as the kidney, liver and lungs, also eventually wear out and must be continually replaced during normal aging. In addition, we sustain injuries in the form of cuts, abrasions, bruises and broken bones. Here, not only must damaged or dead cells be replaced, but new cells must migrate into the damaged region, repair the tissue architecture and restore function. Alternatively, the damaged tissue can be “patched up” and regions containing dead cells replaced with a cell-derived “bandaid” called scar tissue.
These remarkable feats are accomplished, in large part, through the activity of a relatively small number of widely-scattered, self-renewing cells called stem cells. These cells have the ability to proliferate as needed and give rise to rapidly dividing progenitor cells whose progeny subsequently develop into specific lineages of differentiated cells.
Embryonic stem cells (ESCs), derived from early human embryos, have an apparently unlimited growth potential and capacity to differentiate into almost any cell type, but their bane is a tendency to unpredictably develop into cancers in vivo — a property that may seriously limit their safe clinical use. By contrast, adult stem cells, such as mesenchymal stem cells (MSCs), avoid ethical concerns, but these cells are more difficult to propagate in vitro and show a more restricted differentiation potential than ESCs. MSCs can be isolated from umbilical cord blood and, as described in more detail by my collaborator, Dr. Biaggio Saitta, in this issue, can differentiate into bone, cartilage, muscle and connective tissues.
Our work on MSCs, which is currently funded by the New Jersey Commission on Science and Technology, is being done through a collaboration between my laboratory at UMDNJ in Stratford and those of Drs. Biaggio Saitta, Margaret Keller and Jay Leonard at the Coriell Institute. Our goal is to evaluate the genomic stability and differentiation potential of MSCs during and after their preparation for clinical use — in other words, to make sure that treatment of patients with MSCs is safe.
MSCs are extremely rare (only 1-3 per 10,000 cells). This requires them first to be isolated from surrounding cells and then coaxed to undergo extensive proliferation in a cell culture environment (ex vivo expansion) in order to generate numbers of these cells that are clinically useful. However, unlike ESCs, MSCs grown in culture undergo “in vitro aging” — they are subject to the Hayflick limit of roughly 50 mitotic divisions and then lose their ability to replicate further, achieving a quiescent state referred to as cell senescence. Since a major trigger that drives cells into senescence is DNA damage, this suggests that MSCs in long-term cell cultures are becoming progressively more genomically unstable — of course, an undesireable feature for cells that we intend to inject into patients. One type of DNA damage that potently induces senescence is the aging-dependent shortening of telomeres. Telomeres are long stretches of DNA that cap the free ends of chromosomes and are now thought to serve as the “cell aging clock.” They shorten with each mitotic division – making our telomeres progressively shorter as we age. Eventually, one or more telomeres become critically short and dysfunctional, and these are recognized as DNA damage. Attempts to repair such damage can lead to chromosome end-to-end fusions, which disrupt chromosome segregation during subsequent mitoses and cause genomic instability, including chromosome breakage and numerical aberrations (aneuploidy) that are potentially tumorigenic.
Unfortunately, there is a danger that by the time sufficient numbers of MSCs have been generated for infusion into patients, the cell population may contain a significant number of senescent cells as well as some genomically unstable cells that have somehow managed to bypass senescence. Although the fate of these cells after injection into patients is unknown, a recent study has shown that human MSCs derived from adipose tissue can undergo spontaneous transformation after long-term culture and may be prone to malignant transformation. This same phenomenon has been observed in mouse MSCs. These findings emphasize the need for accurate studies that will document the bio-safety of these cells before their infusion into patients, especially in immune-compromised patients whose status might favor the development of tumors.
The goal of our collaborative study is to gain a thorough understanding of how ex vivo expansion of MSCs can affect their stemness (i.e., ability to act as stem cells), genomic stability, and proliferation and differentiation capacity. We are evaluating the genomic stability of MSCs at selected intervals during ex vivo expansion. This evaluation includes identification of abnormal and senescent cells and determination of chromosomal abnormalities in MSCs by G-banding, fluorescence in situ hybridization using chromosome-specific paint probes and spectral karyotyping (SKY). In addition, the effects of prolonged culture on telomere length at the single cell and individual telomere level in MSCs will be determined by quantitative image analysis. We are also investigating the effects of ex vivo expansion of MSCs on their chromatin organization. We predict an enrichment of acetylated histone H3 (acH3) on the proximal promoter regions of genes involved in maintaining the stem cells in their undifferentiated state (called “stemness genes”). We are testing whether MSCs at later culture passages show a reduction in the number of stemness gene promoters that show acH3 enrichment, and are also checking to see if these epigenetic changes coincide with alterations in the differentiation potential of MSCs and expression of their stemness genes. Lastly, it is essential to make sure that MSCs, after completing extensive growth in cell cultures, still retain their proliferation and differentiation capacity. To this end, we are measuring the ability of single MSCs to proliferate and form cell colonies and testing their ability to differentiate toward adipogenic, osteogenic and chondrogenic lineages. Our goal is to shed some light on genetic and phenotypic changes that occur in MSCs during their ex vivo expansion. Once any undesireable changes are identified, it may be possible to sidestep these changes by altering the ex vivo expansion procedures, thus further ensuring the safety and effectiveness of MSCs for therapeutic use.
Robert Nagele is a professor in the Department of Medicine and New Jersey Institute for Successful Aging at the UMDNJ -School of Osteopathic Medicine (SOM). He received his PhD in 1980 from Rutgers University and did his post-doctoral training at Rutgers Medical School. He joined the faculty at SOM in 1983. His research interests are focused on the mechanisms of aging-associated diseases, especially Alzheimer’s disease and cancer. His work in these areas was funded by the Alzheimer’s Association, The Governor’s Council on Autism, the Foundation of UMDNJ and the New Jersey Commission on Science and Technology.