How to Understand Regenerative Medicine - What is It?

There is still much to learn about the optimal treatment paradigm in regenerative therapies including indications, technique, route, dose, timing, and frequency. In order to fully assess therapies, the treating clinician must have a solid understanding of regenerative medicine techniques. Author’s address: Large Animal Surgery, Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843; e-mail: awatts@cvm.tamu.edu.

1. Introduction

Regenerative medicine is the process of harnessing natural healing processes to improve upon tissue repair for a more functional healed tissue. The holy grail of regenerative medicine would be to recapitulate development, resulting in healed tissues that cannot be distinguished from uninjured tissue. Although to date this has not been achieved in musculoskeletal tissues, the potential to substantially improve outcomes with regenerative techniques is considerable. Subsequently, there has been much activity in research and widespread clinical use of regenerative therapies for equine orthopedic applications. Some of the tools for regenerative medicine in orthopedics include stem cells, platelet rich plasma, autologous conditioned serum, growth factors, and gene therapy. Regenerative therapies can be applied by intralesional, perilesional, intraarticular, or intravenous injections.

2. Stem Cells—Defined

Stem cells, unlike their somatic cell counterpart, are self-renewing, highly proliferative, and capable of multilineage differentiation. The ultimate stem cell is made at conception. After fertilization, the zygote consists of totipotent stem cells that are able to form all 3 germ layers as well as placental tissue. Once the zygote becomes a pre-implantation blastocyst, the inner cell mass consists of pluripotent stem cells that will give rise to all 3 germ layers: the ectoderm, mesoderm, and endoderm and can no longer form placental tissues. At this stage the stem cells are embryonic. After day eight, the cells will become either somatic cells (terminally differentiated) or stem cells committed to a specific lineage (multipotent). Subsequently, the stem cells are considered adult-derived, despite their presence in fetal tissues. Local niches of lineage committed multipotent stem cells remain in adult tissue throughout life for normal tissue remodeling and repair. With increasing age, the number, expansion potential, differentiation potential, and socalled potency of stem cells declines; therefore, there is increasing interest in allogeneic embryonic and fetal derived stem cells as well as banking of autologous stem cells from post-natal samples. 

Modifications to these classifications of stem cells (embryonic and adult-derived) are also being investigated and will be briefly discussed. One modification is a fetal-derived stem cell that has been manipulated in vitro to act more like an embryonic stem cell. One such product has been developed and tested in the horse but clinical availability is pending FDA approval.¹ An important benefit of this type of stem cell is its immediate availability as an ‘off-the-shelf’ product and its increased potency due to a pluripotent-like (embryonic-like) state. Another modification is the induced pluripotent stem cell, where in vitro manipulations are applied to adult somatic cells, such as skin fibroblasts, to de-differentiate them and induce a stem cell-like state. The induced pluripotent stem cell is currently being investigated by several equine research groups.

Because of their broad overlap with other cell populations, mesenchymal stem cells (MSCs) cannot yet be accurately sorted by cell surface markers. Therefore, many labs select and isolate MSCs by expanding the tissue culture plastic adherent population of colony forming cells. This translates to a culture period of 2 to 3 weeks, in vitro, to isolate and expand MSCs from clinical samples for autologous therapy. In the horse, MSCs have been isolated from bone marrow, adipose, tendon, muscle, umbilical cord blood and tissue, gingiva and periodontal ligament, amniotic fluid, and blood (Figs. 1–3). The different tissue sources vary in the ease of harvest, expansion potential, and differentiation capacity. Several academic and commercial laboratories provide for the isolation, expansion, and cryopreservation of stem cells from several different tissue sources; namely, bone marrow, fat and umbilical cord, or blood. Directions for collection and shipping procedures are available from each lab. To date, bone marrow derived MSCs from both the horse and human have been the most thoroughly studied and have the most evidence for the ability to undergo chondrogenesis, tenogenesis, and osteogenesis and might contribute to cartilage, tendon, and bone repair as well as modulate inflammation and soft tissue repair within the joint.
 

3. Stem Cells—Autologous or Allogeneic

Autologous (self) therapy has been used most in horses to date. Autologous cells are considered safe with minimal risk for disease transmission. A major disadvantage of autologous cells is that unless cells have been banked prior to injury, their use dictates a delay of 2 to 3 weeks for isolation and expansion. Although many labs are offering banking of autologous MSCs, the long-term viability of cryopreserved MSCs has not been fully elucidated. One way to avoid the culture delay for autologous MSCs is to use patient side kits to concentrate stem cells.² Several commercial kits are available that enrich for the nucleated cellular portion resulting in a higher concentration of MSCs in a small volume from bone marrow and fat. Another method to avoid delay would be to use allogeneic (nonself) cells. Because MSCs are immune privileged, allogeneic cells can be used in nonrelated individuals and without immune testing. Although this has been demonstrated in most species, it has not yet been thoroughly reported in the horse. Use of an allogeneic stem cell line would allow use of an ‘off-the-shelf’ stem cell product and would have several advantages: reduced variability, allow same day treatment, allow use of younger stem cells, and use in aged horses. Finally, it may reduce costs. [...]