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The confusion surrounding stem cells starts at the root, with the most basic but difficult-to-answer question, "What is a stem cell?" Get answers to this and other questions related to stem cell research and therapies.
Stem cells are an exciting biological technology for scientists, engineers, and physicians, but more significantly, stem cell therapies are a source of hope for millions of ailing patients. An SBE Supplement in the Nov. 2010 issue of CEP explored stem cell engineering, including the use of adult stem cells for skeletal tissue engineering applications (1). Despite their promise for this application and numerous others, there is confusion in the field, and in the general public, over the science, medicine, bioethics, and governmental regulation behind this technology.
This article attempts to end this confusion, and discusses the state of various stem cell technologies, including the regulations that govern them and their translation to human clinical therapy.
What are stem cells?
The term stem cell is a generic term to describe a primitive cell that has the ability to self-replicate and differentiate or mature into a less-primitive cell. However, amid the public's growing exposure to news headlines about stem cells, the term has come to be used to describe any cell that gives hope of a cure or treatment. As a result, what is considered a stem cell is a philosophical idea and consequently has been greatly debated among scientists.
Scientists classify cells based on their differentiation potential, starting with a zygote - the most primitive mammalian cell - that forms after the fusion of a sperm and egg. As the zygote becomes an embryo, it is referred to as a blastocyst, which contains many cells called blastocytes. These cells are pluripotent, which means they have the capacity to differentiate into any tissue-specific cell type of an organism.
Embryonic stem cells (ESCs) are derived from blastocytes and are usually harvested from excess in vitro fertilized eggs that are destined to be disposed of once unfrozen. This process has raised well-documented ethical questions related to the destruction of potential life versus the potential to treat terminal illnesses.
ESCs receive biochemical cues from the mother's womb that direct their growth, division, and maturation into lessprimitive cell types. Their capacity for pluripotency makes ESCs potentially useful in the treatment of degenerative diseases - particularly neural-related conditions, including Parkinson's, amyotrophic lateral sclerosis (ALS) (i.e., Lou Gehrig's disease), multiple sclerosis (MS), and spinal cord injuries. Because ESCs are derived from donor tissue rather than the patient being treated, they are classified as allogeneic cells.
As cell maturation continues during pregnancy and after birth, ESCs are replaced with less-primitive adult stem cells (ASCs) that maintain and regenerate various bodily tissues. The two most researched and understood ASCs are hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). HSCs are predominantly located in the bone marrow stromal tissue, and differentiate into blood and immune cell types. They are present in umbilical-cord blood and in peripheral blood at small concentrations. These cells comprise the immune system, and because immune response is important throughout a lifetime, are present in the bone marrow at high concentrations regardless of age.
Certain diseases, radiation, or chemotherapy treatment can ablate HSCs and progenitor cells and leave individuals without a functional immune system. In these situations, doctors employ bone marrow or cord-blood transplants to infuse the patient with healthy HSCs and re-establish the bone marrow. Transplants of this kind have been done for several decades - even before the blood cell lineage and existence of HSCs were fully understood - and represent the earliest use of stem cells as a treatment.
MSCs were first discovered after an in vitro culture of bone marrow grew cell colonies that were able to regenerate bone, hematopoietic, and stromal tissues in vivo. MSCs were later discovered to be present in many skeletal and connective tissues, including bone, muscle, skin, fat, umbilical cord, and placental tissues. Additional research has concluded that MSCs are likely a subset of pericytes - the contractile cells that line the outer surface of blood vessels (2). Although it was originally hypothesized that MSCs contribute directly to healing by differentiating into cells of the damaged tissue type, scientists now believe that the majority of MSCs' effects are paracrine, and that they secrete signaling proteins (called cytokines) that promote other cells to multiply, migrate, differentiate, or change their own protein expression (3, 4). Like HSCs, MSCs are present at high concentrations in vascular tissues regardless of age.
Although HSCs and MSCs have the ability to regulate and regenerate tissues that naturally undergo remodeling or turnover, or are frequently injured by trauma, their potential to regenerate whole organs (e.g., liver, kidneys, neural tissue, etc.) has not been established. In an attempt to harness the pluripotent potential of ESCs in an autologous (i.e., cells harvested from the patient) manner, research was conducted to create induced pluripotent stem (iPS) cells through a molecular process that reactivates a set of critical embryonic-stage genes in adult skin cells (5). These iPS cells have demonstrated the potential to differentiate into tissue types of all three skin germ layers. Figure 1 indicates where iPS cells fit into the cell hierarchy and differentiation potential structure.
How are stem cells regulated?
In the U.S., the Food and Drug Administration (FDA) regulates stem cells under 21 Code of Federal Regulation (CFR) 1271, which applies to human cells, tissues, and cellular and tissue-based products (HCT/Ps). If cells and tissues are only minimally manipulated (e.g., culture-expanded, cryopreserved, harvested by enzyme digestion, etc.), if they are used homologously (in a form in which they were harvested from the body [a somewhat ambiguous definition]), and if they are derived from the patient or a first- or seconddegree blood relative, they are regulated under Section 361 of the Public Health Service Act. If those conditions are not met, cells and tissues require an investigational new drug exemption by the FDA, a biologic license application, and clinical trials before they can be marketed and used to treat patients, and are regulated under Section 351.
The only type of stem cell treatments available in the U.S. without a drug trial are those that utilize the patient's own stem cells, harvested and administered in a single surgical event, and with no more than minimal manipulation. Several companies market devices to concentrate stem cells from bone marrow aspirate within the requirements of Section 361. Since 2001, these devices have been employed in orthopedic procedures, including spine, knee, hip, and rotator cuff surgeries, and for the treatment of osteoarthritis, degenerative disc disease, and nonhealing wounds and ulcers.
Although it is a common misconception that the use of all stem cells is illegal in the U.S., these devices and procedures are currently available clinically. No type of stem cell research, including ESC research, is or has ever been illegal in the U.S.; however, federal research funding allocations have shifted periodically with election cycles.
Allogeneic-cell products, including those derived from cadavers or amniotic tissue, do not qualify as Section 361 products if they contain metabolically active (i.e., living) cells that are not from the patient or an immediate relative. ESCs are not covered by Section 361, because they are harvested from a donor, are more than minimally manipulated (they are grown in a lab and frozen), and are used for nonhomologous applications. Although iPS cells are autologously derived, they require drug trials for many of the same reasons that EPS cells do.
Umbilical-cord-blood storage has been popular for many parents since the 1990s as a precautionary measure. But because the stem cells in cord blood are primarily HSCs, cord blood banking is only advisable for families with a history of blood diseases (e.g., leukemia). Although the cord blood is autologous (or first-degree in the instance of siblings), the necessary freezing, thawing, and cultureexpansion processes represent more than minimal manipulation. Therefore, the clinical use of cord blood can only be performed with an exemption from the FDA, and is examined on a case-by-case basis.
Over the past two decades, numerous companies have developed methods to isolate autologous stem cells. Many of these technologies harvest bone marrow or adipose tissue and separate them by means that are common laboratory practices, but are considered beyond minimal manipulation by the FDA. These methods use enzymes or mechanical means to break up tissue samples to release the stem cells, multiplying them through in vitro culture expansion or purifying them from a heterogeneous population by chemical or biomarker separations.
The FDA has issued warning letters and cease-anddesist orders to such companies. One Colorado company developed a method for expanding a culture of a patient's bone marrow MSCs to treat various diseases. The company manipulated the culture because the number of stem cells present in fresh bone marrow aspirate was deemed to be insufficient. Although the procedure was performed completely within the state of Colorado, which would seem to negate the need for federal intervention, the federal courts ruled that the FDA had the authority to regulate the use of patients' cells if they were not compliant with the rules of Section 361. (In this case, the in vitro culture expansion of cells went beyond minimal manipulation.) The founders of the company have since opened a clinic in the Cayman Islands, where they perform their procedures outside the jurisdiction of the FDA.
Has there been any clinical success?
Beyond the successful history of bone marrow transplants, other clinical studies have demonstrated remarkable outcomes utilizing autologous stem cells. Since 1990, statistically significant improvements in patients with nonhealing, nonunion fractures of leg bones and avascular necrosis of the hip, as well as drastically improved outcomes in the surgical repair of rotator cuff tears, have been realized with the use of bone marrow concentrate (BMC), as opposed to surgery that does not utilize this technology (6-8).
In a degenerative disc study conducted in 2014, patients with severe low-back pain who received injections of autologous BMC experienced more than a 60% reduction in pain over a year's time (9). The most intriguing aspect of this study is the observed correlation between MSC concentration in the BMC and improved patient outcomes. This indicates that physicians should consider increasing MSC concentration during harvesting and processing of a patient's cells.
Beyond orthopaedic applications, autologous stem cells have been used successfully in the treatment of third-degree and radiation bums, spinal fusion, spinal cord injuries, cardiac repair after heart attack, MS, and Parkinson's disease. But because there is relatively negligible patentable intellectual property for minimally manipulated autologous cells, companies and investors have little motivation to fund expensive clinical trials that would also benefit their competitors.
What are the risks?
While stem cells offer unique treatment options for devastating conditions, their safety and risk varies among the different types of stem cells. For example, ESCs receive specific signaling in the womb to trigger growth and differentiation; however, when they are injected into adult recipients, there is a high rate of tumor or cyst formation (10). This phenomenon also occurs with iPS cells, which have given rise to teratoma formation in as many as 50% of recipients (11). This suggests a link between pluripotency and tumorigenicity, and requires the predifferentiation of ESCs and iPS cells into defined tissue lineages prior to implantation, which remains a challenge.
A company that developed an allogeneic MSC product focused clinical trials on autoimmune disorders, including graft-versus-host disease (GvHD) and Crohn's disease. Of the patients in the initial GvHD trial, 50% experienced mild to moderate adverse effects (e.g., antidonor antibody production, fever, etc.) (12).
Another company developed a proprietary formulation of allogeneic MSCs marketed for use in spinal fusion, intervertebral disc injections, and cardiovascular treatment after heart attacks. The cardiovascular clinical trial reported that 13% of patients in the high-cell-dose group produced chronic antibodies against the donor cells (13).
This and similar studies revealed an inverse relationship between allogeneic cell dose and treatment effectiveness. This may be a consequence of the unnaturally high concentrations of MSCs (several thousand times greater than physiological levels in similar tissue) or the high number of foreign cells (which can incite an increased immune response).
Final thoughts
Stem cell therapies will undoubtedly be an integral part of regenerative medicine, and will play a major role in future clinical therapy. Some autologous stem-cell products available to physicians and patients today have demonstrated promising efficacy, with no evidence of safety risks. Other technologies that use manipulated, allogeneic, and/or embryonic stem cells show promise, but will require additional research and cooperation between scientists, biotechnology companies, and regulatory agencies.
The trade-offs between potential and demonstrated effectiveness, risks to patients, cost of therapy, and regulatory environment must be considered by patients, physicians, scientists, biotechnology companies, and government regulatory agencies for all stem cell therapy options (Table 1 ).
The public knowledge and subsequent demand for stem cells has never been greater, but government regulation and unproven safety (primarily for ESCs and iPS cells) have hindered private sector investment in the industry. Scientists and physicians need to continue to gather and report data, and work with industrial, academic, insurance, and regulatory entities to advance stem cell treatments for the benefit of patients. ^^3
LITERATURE CITED
- 1. Murphy, M. B., et aL, "Engineering a Better Way to Heal Broken Bones," Chem. Eng. Progress, 106 (11), pp. 37-43 (Nov. 2010).
- Caplan, A. I., "All MSCs are Pericytes?," Cell Stem Cell, 3 (3), pp. 229-230 (Sept. 2008).
- Caplan, A. I., and D. Correa, "The MSC: An Injury Drugstore," Cell Stem Cell, 9 (1), pp. 11-15 (July 2011).
- Murphy, M. B., et aL, "Mesenchymal Stem Cells: Environmentally Responsive Therapeutics for Regenerative Medicine," Experimental and Molecular Medicine, 45 (11 ), DOl: 10.1038/ emm.2013.94 (Nov. 2013).
- Takahashi, IC, and S. Yamanaka, "Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors," Cell, 126 (4), pp. 663-676 (Aug. 2006).
- Hernigou, P., et aL, "Percutaneous Autologous Bone-Marrow Grafting for Nonunions," Journal of Bone and Joint Surgery, 87 (7), pp. 1430-1437 (July 2007).
- Hernigou, P., et aL, "Percutaneous Implantation of Autologous Bone Marrow Osteoprogenitor Cells as Treatment of Bone Avascular Necrosis Related to Sickle Cell Disease," The Open Orthopedics Journal, 2, pp. 62-65 (2008).
- Hernigou, P., et aL, "Biologic Augmentation of Rotator Cuff Repair with Mesenchymal Stem Cells During Arthroscopy Improves Healing and Prevents Further Tears: A Case-Controlled Study," International Orthopeodics, 38 (9), pp. 1811-1818 (Sept. 2014).
- Pettine, K. A., et aL, "Percutaneous Injection of Autologous Bone Marrow Concentrate Cells Significantly Reduces Lumbar Discogenic Pain Through 12 Months," Stem Cells, DOl: 10.1002/ stem. 1845 (Sept. 2014).
- Knoepfler, P. S., "Deconstructing Stem Cell Tumorigenicity: A Roadmap to Safe Regenerative Medicine," Stem Cells, 27 (5), pp. 1050-1056 (May 2009).
- Yamanaka, S., "A Fresh Look at iPS Cells," Cell, 137 ( 1 ), pp. 13-17 (Apr. 2009).
- Onken, J., et aL, "Successful Outpatient Treatment of Refractory Crohn's Disease using Adult Mesenchymal Stem Cells," presented at the American College of Gastroenterology Annual Meeting, Las Vegas, NV (Oct. 2006).
- Perin, E. A., "Phase II Dose-Escalation Study of Allogeneic Mesenchymal Precursor Cells in Patients With Ischemic and Nonischemic Heart Failure," Circulation, 124, pp. 2365-2374 (Nov. 2011).
RACHEL M. BUCHANAN
THE UNIV. OF TEXAS AT AUSTIN
DANIEL BLASHKI
THE UNIV. OF MELBOURNE
MATTHEW B. MURPHY
CELLING BIOSCIENCES Copyright: (c) 2014 American Institute of Chemical Engineers
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