PHYSIOLOGICAL CHANGES: STEM CELLS

The process of aging is controlled by a variety of cellular and molecular mechanisms. This process is a continuum of events that are initiated during embryonic development, are genetically determined, and continue throughout all of life. There is a genetic program that controls how and where arms and legs form in the embryo, how much these appendages grow after birth and how much bone will be lost as a person becomes older. Interestingly, old in this context means "after skeletal maturity," which occurs in a person's early thirties. Assuming this genetically controlled continuum between embryology and the aging process, it is important to understand the process of tissue formation, the process of tissue expansion, and how tissue is maintained throughout life. Considered here is a class of tissues that form from the middle layer of the three-layered embryo, the mesodermal layer ; this class of tissue is summarily referred to as mesenchyme, and includes bone, cartilage, muscle, tendon, ligament, fat, and other connective tissues.

Embryonic development and mesengenesis

The mesodermal layer in the embryo is composed of cells that will divide many times as an organism grows, and the cellular descendants will eventually differentiate into unique cell types that fabricate very diverse mesenchymal tissues. Thus, from a uniform population of mesenchymal cells, unique differentiation pathways give rise to mesenchymal tissues which differ greatly in morphology and function. In addition, these tissues have unique shapes and unique chemistries. The important aspects of the complex molecular signaling that determines the shapes and sizes of various tissues will not be considered here, but the concept of a multichoice differentiation pathway—for example, bone versus cartilage—is essential to understand with respect to neonatal and adult life.

In the embryo, as in the adult, a common mesenchymal progenitor cell gives rise to either bone or cartilage. This cell could also become any other differentiated mesenchymal cells, and is thus called a mesenchymal stem cell. The local molecular microenvironment controls whether mesenchymal stem cells divide, whether they go down one differentiation pathway or another (e.g., bone versus cartilage), whether they are quiescent, or whether they expire. This global scheme has been called mesengenesis, and it is pictured in Figure 1, with the mesenchymal stem cell at the top and each differentiation pathway separated and depicted in an over-simplified sequence of lineage steps. The molecules and microenvironments that control these events in the embryo and in the adult are not fully known.

Adult tissues

Skeletal maturity is observed in the early to mid-thirties in modern, well-nourished humans. However, the process of growth that has occurred into the thirties from embryology occurs on a backdrop of continued tissue replacement or rejuvenation, referred to as tissue turnover. In bone, for example, there is a constant destruction of bone tissue followed by refabrication of the lost bone to the extent that the entire skeleton is replaced every two to ten years, depending on the age of the individual. This is in addition to the continued growth or expansion of bone that occurs from birth through the teenage years. Thus, in early life, the process of bone formation far exceeds the process of bone loss. Importantly, osteoblasts, the bone-forming cells, form sheets of many cells that fabricate layers of new bone. The extent of such bone formation is directly related to how many osteoblasts are in this formative sheet. These osteoblasts also have a genetically fixed life span—a week to two weeks for most humans. Thus, while sheets of osteoblasts are fabricating bone in various locations within the body, some osteoblasts are dropping dead. Their place in the fabrication sheets is taken by newly born osteoblasts—it takes two or three generations of osteoblasts to fill the holes in the bone formed by the natural bone-destruction process. Therefore, mesenchymal stem cells must be present in sufficient numbers to provide descendants that traverse the differentiation pathway to become newly born osteoblasts. In adults, these mesenchymal stem cells are in the bone marrow in close proximity to bone and are associated with blood vessels that nourish bone.

Another way of stating that skeletal maturity peaks in a person's thirties is to say that the process of bone destruction becomes equal to the process of bone fabrication. As a person progresses in age past the thirties, the destructive process exceeds the formative events, resulting in cumulative bone loss, which is referred to as osteoporosis when the bone structure becomes fragile and susceptible to fracture. Although osteoporosis is considered a disease of the aged, the extent of bone loss and severity is dependent on the bone stock present in a person's thirties and the relative balance of bone formation versus destruction thereafter. Clearly, a key variable in this complex process is the number of mesenchymal stem cells in a particular location—and the number of their progeny that are cued into the bone-forming pathway at any one time.

Bone repair

In adults, when a bone breaks, the repair process involves a dramatic shift in the balance of bone formation compared to bone destruction toward a process that strongly favors bone formation. The bone repair process involves the flooding of the break site with mesenchymal stem cells, which span and connect the broken ends of the bone. If the broken ends are stabilized by a physician or by nature, the mesenchymal stem cells differentiate directly into bone-forming cells (osteoblasts) that are oriented into fabrication sheets by the neighboring blood vessels. If the break is not stable, the mesenchymal stem cells differentiate into a spanning plug of cartilage and connective tissue that serves to stabilize the break site, allowing an outer layer of bone to form around the plug bridging the broken ends of the bone; this bone-cartilage composite tissue is called the repair callus. The cartilage on the inside of the callus further develops into what is called hypertrophic cartilage, and these individual end-stage cartilage-producing cells expire.

As the hypertrophic chondrocytes are expiring, they produce and release chemicals that eventually signal blood vessels and new mesenchymal stem cells to enter this site. This combination of new mesenchymal stem cells and blood vessels creates the microenvironment for the formation of bone where cartilage had previously been located. These events have led to the erroneous generalization that cartilage is replaced by bone, when actually cartilage is replaced by blood vessels and a new batch of mesenchymal stem cells, which later form bone. Eventually, the callus is remodeled and sculpted so that scarless bone is located at the site of the original break and is wholly integrated into the pre-existing bone.

The speed at which these repair events occur is directly dependent on the number of mesenchymal stem cells that come to reside in and form the initial callus. It is observed that a person ten years of age and a person ninety years of age repair bone breaks at different rates. One cause for these rate differences is that the number of mesenchymal stem cells in the vicinity of the bone breaks is vastly different. Thus, bone formation, bone growth, bone homeostasis, and bone repair are all dependent on the number and receptive signaling capacity of mesenchymal stem cells.

Mesenchymal stem cell numbers

Mesenchymal stem cells are in bone marrow, are associated with blood vessels, are present in the connective tissue of muscle, and are in a number of anatomical locations. Because mesenchymal stem cells are located throughout the body, it is currently impossible to accurately determine the total number that exist during life. One approach to providing an estimate is to determine the number of mesenchymal stem cells in a standard portion of bone marrow. A number of laboratories have attempted to provide such estimates throughout life. At the Skeletal Research Center, at Case Western Reserve University, scientists isolate and purify mesenchymal stem cells and encourage them to attach to and grow on petri dishes, in which the cells form colonies, each of which is derived from one cell. They have developed the technology for isolating, purifying, and expanding mesenchymal stem cells in culture dishes without the mesenchymal stem cells entering any of the differentiation pathways. Based on this technology, Skeletal Research Center scientists have processed fresh bone marrow specimens from hundreds of individuals ranging in age from newborns to people in their eighties and nineties. The rate of cell division and the sensitivity to agents that cause differentiation into the mesengenic lineage pathways appear to be identical among all of these cell preparations, independent of the age of the individual providing the bone marrow specimen. Thus, young and old mesenchymal stem cells appear to be identical in cell culture.

Again, emphasizing that colony counts per sample of bone marrow are crude estimates of the frequency of mesenchymal stem cells in marrow, it is clear that large age differences in the numbers of mesenchymal stem cells exist, from very high levels in newborns (about one in ten thousand nucleated marrow cells) to levels tenfold to one hundred-fold less in older individuals. Thus, when a frail eighty-year-old woman breaks a bone, the slowest step in the repair process is the accumulation of enough mesenchymal stem cells at the break site to form a proper callus. When bone is fabricated around the callus to bridge the broken ends, this bone forms, per osteoblast, about as rapidly as in younger individuals. Thus, both mesenchymal stem cell and osteoblast functioning in older individuals appear to be quite similar; what is quite different is the distribution of, and probably the total number of mesenchymal stem cells available to the repair site. This is in contrast to the number of hematopoietic progenitor or stem cells, which stays relatively constant throughout life at about one per ten thousand nucleated cells. Thus, the process of aging appears to involve a decrease in the number of responsive mesenchymal stem cells, not a decrease in function.

In the context of evolution, those individuals who can maintain the highest concentrations of mesenchymal stem cells throughout life will enjoy a selective advantage with age. This advantage involves the rate of tissue repair (as in the case discussed above for bone), and also is related, in part, to the balance between tissue formation and tissue turnover events. The thesis advanced here is that the embryo has the highest tissue levels of mesenchymal stem cells, and that these levels decline with age. The individuals who can maintain high mesenchymal stem cell levels will be able to maintain high tissue fabrication levels and more effectively balance the natural destructive activities observed in almost every mesenchymal tissue. Since both muscle tissues and the dermis of skin are derived from the same small sectors of somites in embryos, it is interesting to note that the loss of muscle mass with age is correlated with texture changes in the skin. One wonders whether this is because both dermal and muscle cells are on the same biological clock, or because the local titers of mesenchymal stem cells, or their reactivity, is decreasing with age.

Mesenchymal stem cells and future aging therapies

If one of the major causes of mesenchymal tissue aging is the decreased availability of mesenchymal stem cells, an obvious quick-fix would be to obtain some marrow, and then isolate and culture-expand the mesenchymal stem cells. It has been shown that mesenchymal stem cells can be culture-expanded one billion-fold without affecting their differentiation potential. With this large number of cells, it would seem possible to merely add these cultured mesenchymal stem cells back to blood-stream marrow and other sites serviced by the blood supply, and thereby raise the body level of mesenchymal stem cells. However, although some mesenchymal stem cells that are put back into the blood stream do make it back into the marrow compartment, relatively few do so. Consequently, increasing the body's load of mesenchymal stem cells requires understanding how the cells target to particular locations along the blood-vessel pathways, and then to efficiently transport the cell through the wall of the blood vessel into the tissue. Alternately, by genetically adjusting the mesenchymal stem cells in culture to fix or replace mutated genes, it may be possible to improve the fabrication capacity of the differentiated progeny to maintain tissue integrity and counteract aging-related tissue destruction. Even if these therapeutic approaches fail, it is clear that management of mesenchymal stem cells can have profound effects upon the aging process.

Arnold I. Caplan

See also Blood; Cellular Aging; Cellular Aging: Cell Death; Osteoporosis.

BIBLIOGRAPHY

Caplan, A. I. "Mesenchymal Stem Cells." J. Ortho. Res. 9 (1991): 641–650.

Caplan, A. I. "The Mesengenic Process." In Clinics in Plastic Surgery 21 (1994): 429–435.

Haynesworth, S. E.; Goldberg, V. M.; and Caplan, A. I. "Diminution of the Number of Mesenchymal Stem Cells as a Cause for Skeletal Aging." In Musculoskeletal Soft-Tissue Aging: Impact on Mobility. Edited by J. A. Buckwalter, V. M. Goldberg, and S. L-Y. Woo. Rosemont, Ill.: American Academy of Orthopaedic Surgeons, 1994. Pages 79–87.

Haynesworth, S. E.; Reuben, D.; and Caplan, A. I. "Cell-based Tissue Engineering Therapies: The Influence of Whole Body Physiology." Advanced Drug Delivery Review 33 (1999): 3–14.

PICK'S DISEASE

See Fronto-temporal dementia