Abstract
Not too long ago, several motivated and forward-looking articles were published describing the cellular and molecular properties of mesenchymal stem cells (MSCs), specially highlighting their potential for self-renewal, commitment, differentiation, and maturation into specific mesoderm-derived lineages. A very influential publication of that period entitled “Mesenchymal stem cells: No longer second class marrow citizens” [1] raised the point of view that “…challenges to harness MSC cell therapy to treat diseases … need to wait for the full comprehension that marrow is a rich source of mesenchyme-derived cells whose potential is still far from fully appreciated.” Whether or not the prophecy of Gerson was fulfilled, in the last 8 years it has become evident that infusing MSCs into patients suffering a variety of disorders represents a viable option for medical treatment. Accordingly, a vast number of articles have explored the privileged cellular and molecular features of MSCs prepared from sources other than the canonical, represented by the bone marrow. This review will provide more information neither related to the biological attractiveness of MSCs nor to the success after their clinical use. Rather, we would like to underscore several “critical and tangential” issues, not always discussed in biomedical publications, but relevant to the clinical utilization of bone-marrow-derived MSCs.
Clinical Uses of Mesenchymal Stem Cells: Current Conditions Under Treatment
I
MI, myocardial infarction.
Manufacturing of Bone-Marrow-Derived MSCs for Utilization in Clinical Studies
For utilization in clinical studies, MSCs have been prepared from various sources, including bone marrow, umbilical cord blood, adipose tissue, and placenta-derived tissues. However, in the vast majority of clinical trials currently under development, the preferred source of MSCs has been adult bone marrow (Table 1).
The frequency of MSCs in human bone marrow is very low and depending on the age may comprise ∼1 cell for each 10,000, 100,000, or 250,000 bone marrow mononuclear cells [16]. Accordingly, for utilization in a clinical setting, the minute quantity of MSCs obtained after successive bone marrow aspirations needs to be further increased (ex-vivo expansion).
In this vein, several methods have been described [17 –20]; however, ex-vivo expansion, a procedure mainly based on the capacity of MSCs to adhere to plastic surface, stands as the most utilized. Under these conditions MSCs divide several times, whereas free culture surface is available. As soon as the resulting primary monolayer of expanded MSCs becomes semiconfluent, adherent cells are gently dislodged by using either enzymatic or nonenzymatic cell dissociation reagents and the expansion procedure is reinitiated [21 –23].
It is evident that the outcome of any procedure tending to produce enough MSCs to achieve the requirements of a clinical study must be the safekeeping of the biological quality of the resulting cellular product rather than its quantity. Due to the scarcity of data on the attributes of MSCs in the bone marrow (“native” MSCs, Table 2), it has been difficult to establish to what extent ex-vivo expansion procedures modify the biological traits of “native” MSCs.
During ex-vivo expansion, MSCs undergo several population doublings while retaining the typical spindle-shaped morphology, adherence characteristics, immunophenotype, and multilineage differentiation potential. As compared with “native” MSCs, the just mentioned features remain unchanged during early passages (“short-term ex-vivo expanded,” Table 2); however, they are dramatically modified as the passage number increases (“long-term ex-vivo expanded,” Table 2). At these extreme stages of expansion, the resulting cell population (still designated as MSCs) retains morphological and attachment attributes of early passed MSCs; however, it expresses senescence markers and a restricted differentiation potential. The information depicted in Table 2 is only presented as a reference for comparing the features of bone-marrow-derived MSCs (BM-MSCs) after distinctive periods of ex-vivo expansion. It is evident that ex-vivo expansion of BM-MSCs, beyond a certain limit, produces subsets of phenotypically and functionally distinct cells. Probably all these cells fulfill the minimal criteria for defining multipotent mesenchymal stromal cells [36]; however, their clinical potential, in terms of safety and efficacy, may well be restricted [29,37,38].
Limitations to Manufacturing a Cell Product Containing BM-MSCs
Studies have shown that diverse patient's conditions affect either the content of “native” MSCs in the bone marrow or the characteristics of the cell product under manufacturing; accordingly, these observations must be taken into account when developing BM-MSC-based cell therapies, particularly, for autologous use. Among these conditions, the following are relevant and quite recurrent.
Age of the donor. It has been reported that MSCs manufactured from the bone marrow of young donors (18–29 years) as compared with cells prepared from older donors (68–81 years) exhibited a decreased maximal life span, a lower population doubling, and accelerated senescence. Whether age of the donor also affects the differentiation potential of MSCs to adipocytes and/or osteoblasts has not been well established [39 –42].
Alcohol consumption. Cellular studies have demonstrated that ex-vivo-expanded human MSCs exposed to alcohol exhibit a modified commitment and differentiation pathway toward the adipogenic lineage and a concurrent alteration of osteogenic differentiation [43 –46].
Prescription medications. In clinical studies involving the use of autologous MSCs, an adequate registry of various concomitant medications emerges as important due to specific deleterious effect(s) these drugs may have on the differentiation potential of MSCs, as sustained by cellular, molecular, and/or preclinical studies (Table 3).
MSCs, mesenchymal stem cells.
Medical condition. Several distinctive properties of MSCs, like the differentiation potential (mainly into adipocytes and osteoblasts), are severely restricted or modified in patients suffering from various diseases, including osteoporosis [60], alcohol-induced osteonecrosis [61], myelodysplastic syndromes [62], obesity and type 2 diabetes [63,64], and amyotrophic lateral sclerosis [65].
Whether the quality and quantity of MSCs from other sources [66,67] is also affected by age, drugs, or other medical problems in the donor has not been entirely established. In the case of umbilical cord blood (UCB)–derived MSCs, data have shown that the content of circulating UCB-MSCs is higher in preterm babies rather than in babies born at full term [11]. Another interesting feature of UCB-MSCs is that after extensive ex-vivo expansion (up to 15 passages), growth kinetics, immunophenotype, and production of growth factors are being maintained quite stably [68].
Similarly, in adipose-tissue-derived MSCs [prepared either from intact adipose tissue or stromal vascular fraction (SVF)], while many features of MSCs are similar, the differentiation potential is not. MSCs from intact adipose tissue differentiate into osteoblasts and chondrocytes, whereas SVF-MSCs display a unique differentiation pattern to adipocytes [69 –71]. In addition, the medical condition of the donor also affects the properties of MSCs derived from adipose tissues [72].
Autologous or Allogeneic MSCs in Cell Therapies?
The immunological features of MSCs are extremely appealing. Analyses of surface markers have shown that MSCs express MHC class I, but not MHC class II, molecules. Thus, MSCs are protected against deletion by natural killer cells and escape recognition by alloreactive T-cells. In addition, MSCs do not express co-stimulatory molecules (CD40, CD40L, CD80, or CD86) required for effector T-cell induction [14,15,73].
The absence of the co-stimulatory molecules results in T-cell activation (via MHC class I) and thus the genesis of anergic T-cells [74]. Consequently, MSCs evade allogeneic rejection. These characteristics have supported the notion that “allogeneic” and “autologous” MSCs are indistinguishable.
The use of allogeneic MSCs has been considered as a secure option for stem cell therapies wherever the quality and/or quantity of autologous MSCs are jeopardized. In fact, allogeneic MSCs represent a treatment option not only for degenerative diseases, but also for a wide spectrum of other debilitating conditions, including inflammatory, autoimmune, or allograft rejection diseases. Most of these studies have shown, after completion of phase I, that the infusion of allogeneic MSCs is feasible, well tolerated, and with no adverse or serious effects [75 –79].
Nonetheless, the notion that BM-MSCs are immunoprivileged has been reexamined in the last years. In a murine model of allogeneic bone marrow transplantation, it was demonstrated that allogeneic MSCs induce a memory T-cell response resulting in the rejection of the allogeneic graft [80]. In addition, another view of MSC immunoprivilege arose after results showing that a shift from an immunoprivileged to an immunogenic state is closely associated to MSC differentiation potential [81,82].
The notion that allogeneic and autologous MSCs appear to be the same has stimulated the creation of private banks to manufacture and cryopreserve MSCs from healthy third party donors (“off-the-shelf” cells) to treat clinical disorders that require intervention and support at early time points of the disease [83]. In few cases, MSCs are manufactured by well-regulated private institutions and thus the cryopreserved product fulfills the (minimal) biological quality requirements, as established by international committees [36]. This is not the case for a large number of adaptable establishments (most involved in stem cell tourism), where the aim is the production of large amounts of cells under conditions poorly regulated.
MSCs in Cell Therapies: As a Single or a Combination Cell Product?
The translation of the majority of MSC-related studies (molecular, cellular, and preclinical) supports the notion that a “repair cell product” should contain merely a suspension of ex-vivo-expanded MSCs without any other addition. This is the substantiation of the most “canonical” properties of MSCs, which are commitment and differentiation to a number of mesoderm-derived lineages, production/release of growth factors/cellular mediators [84], modulation of the immune response [15,85], deliver trophic support to host cells [86 –89], and, last but not least, the stimulation of endogenous (stem) cells to proliferate [89,90]. As a result, the infusion of this “super repair cell” will engender a clinical improvement in patients suffering a vast array of diseases. This is the case for most of the clinical trials depicted in Table 1 as well as in Table 4 (see single therapeutic product). On the other hand, a number of preclinical and/or clinical studies have hypothesized that an option to augment the clinical efficacy of an MSC-based product could involve the concomitant use of MSCs with another cell type(s), growth factor(s), and/or a signaling/structural protein. As a result, such “combination products” may be proficient in the activation of “distant” cellular or molecular processes, which in conjunction with the “local” effect(s) generated by MSCs might well enhance the repair process. Several of these clinical therapeutic approaches are depicted in Table 4 (see combination therapeutic product).
HA/TCP, hydroxyapatite/tricalcium phosphate ceramic; BMP-7, bone morphogenetic protein 7; PRP, platelet rich plasma; HA, hyaluronic acid; Ca++, calcium; DEX, dexamethasone; EPCs, endothelial progenitor cells; HSCs, hematopoietic stem cells.
Cell Dose and Delivery Routes for an MSC-Based Cell Product
Cell dose
After examining the design and results of the vast majority of clinical studies employing MSCs (most at phase I/II), it is difficult to recognize the rationale for establishing an optimal cell dose to be infused into a patient. In most settings and beyond the nature of the diseases under treatment, the criterion followed to define a cell dose has been based either in the translation of preclinical studies and/or in a dose adjustment with respect to similar trials. In addition, authors have pursued the use of the standardized cell dose (1–5×106 cells/kg body weight) recommended for the infusion of hematopoietic stem cells (“bone marrow” transplants). In not too many studies the design incorporates a double-bind, placebo-controlled, and dose ranging proposal. A noteworthy example of the latter case is illustrated by a few clinical trials aimed to evaluate the clinical effect(s) of MSCs in cardiovascular diseases [77,100,101] and graft-versus-host diseases [76,102].
Delivery route
The success of a clinical protocol utilizing adult stem cells depends on several factors; among them, the choice of a suitable delivery route facilitates the plenteously migration and homing of the “repair” cell into a distinctive site. Despite the enormous attractiveness and credibility adjoining MSC-based therapies, not enough information exists to outline a precise and favorable route(s) for the infusion of the cell product. Animal studies demonstrated that the vascular route was an appealing, noninvasive, prompt, and safe mode of infusing MSCs. Even though after intravenous infusion, a despite that donor cells were found in many mesenchyme-derived tissues of the recipient, a large amount entered and became trapped in the lungs and accumulate there over long periods of time [103 –108]. Regardless of the above evidence, the intravenous infusion of MSCs currently stands as a highly utilized procedure in clinical studies, including critical limb ischemia, acute myocardial infarction (MI), spinal cord injury, multiple sclerosis, and epidermolysis bullosa ([101]; ClinicalTrials.gov Identifier: NCT01351610, NCT00877903, NCT01274975, NCT00813969, and NCT01033552).
In the search for the most effective route for MSC infusion, an appealing example corresponds to those clinical studies assessing improvement in the cardiac function of MI patients, after intracoronary [98,109], intramyocardial [110], or transendocardial ([111]; ClinicalTrials.gov Identifier: NCT00768066, NCT01087996, and NCT00790764) infusion of MSCs. After reviewing these studies and despite results showing that in all cases, MSC infusion is feasible, safe, and devoid of adverse, it is evident that still more information is required to associate efficacy with one or various infusion routes.
In the case of neurological conditions, the noninvasive intrathecal route administration of MSCs to amyotrophic lateral sclerosis patients [91,112 –114], which is safe and tolerable, represents a paradigmatic translation of animal findings into the clinic [115 –117].
Migration and Homing of a Cell Product Containing MSCs to the Desired Injured Site
Migration of MSCs
From the perspective of cell therapy, cell migration is a broad and rather imprecise term describing the relocation of a cell product from the infusion site to the place to be amended or repaired. It is obvious that in this process, specific cellular (cytoskeleton) and molecular signals (extracellular matrix molecules), cell-to-cell contacts, and adhesion ligands and receptors are involved [118].
Precise mechanisms underlying migration of MSCs into a specific site(s) are still not known. Cellular studies have shown that MSC migration to a final microenvironment or niche appears to be a cell-membrane-associated process. This is supported by data showing that changes in specific MSC-membrane antigens (like modification of antigen CD44), as well as activation of the non-neural cholinergic system (via nicotinic acetylcholine receptors), are involved in MSC migration [5,119 –121]. Whether other cellular and molecular mechanisms are also involved in MSC migration and homing is not yet fully comprehended.
In this context it is evident that more research is required to understand (1) to what extent MSC ex-vivo expansion and/or other manufacturing-related methods (i.e., preparation of cells for infusion and/or cryopreservation and addition of preserving/storage agents) affect/modify MSC migration and (2) whether the “receptive” microenvironment(s) of the tissue aimed to be “repaired” has been modified by the injury, disease, or other factors [122 –124].
It is clear that a more comprehensive knowledge of the questions just discussed will permit an all-inclusive translation of cellular and molecular information into the clinical settings [121,125,126]. As anticipated, “…one day we will be able to directly influence the in vivo mechanisms that activate and direct native MSCs towards the sites of inflammation and injury in order to trigger and enhance tissue regeneration…” [5].
Homing of MSCs
A main issue for endorsing the effectiveness of a cellular therapeutic intervention is the assurance that the number and quality of “repair” cells to reach and home into the damaged tissue is satisfactory. On the other hand, and since no cell lives in isolation, low/inefficient homing of infused cells will be a major limitation for stem-cell-based therapies.
Several appealing studies have been performed to better understand the mechanisms involved in MSC homing. Among them, the following findings look relevant in the intent to better comprehend MSC homing: (1) CCR2, a member of the cytokine receptor family, and its ligand MCP-1 are involved in the homing of MSCs into the damaged heart of a model of transgenic mice [127]; (2) the nuclear translocation of CXCR4 upon chemokine stimulation contributes to MSC homing [128]; and (3) factors related to MSC ex-vivo expansion and manufacturing may modulate homing [106,129 –131].
Better knowledge of the mechanisms by which MSCs home to specific sites will permit their intervention prior to infusion (a sort of “clinical homing”), with the subsequent reduction in the number of cells needed to be manufactured (less ex-vivo manipulation), enhancement in therapeutic effect(s), and the accomplishment of the ultimate aim of cell therapy, which is to provide better outcomes to the patient. In addition, the understanding of homing mechanisms and its putative manipulation should have a tremendous impact on the development of “instant stem cell therapies” [124,132].
Evaluation of Safety and Efficacy After Administration of an MSC-Based Cell Product
Several clinical trials have been initiated to explore whether the infusion of autologous or allogeneic MSCs is safe and efficient for the clinical treatment of patients suffering from a vast array of different diseases.
Usually, safety and efficacy evaluation implies the assessment, in a time-dependent manner, of adverse events (rate and grade) and improvement in specific clinical parameters, which may occur immediately after infusion and/or along the follow-up period, as established in the respective protocols.
The choice of proper parameters to assess both safety and efficacy is certainly highly complex and dependent on several factors, like type of disease, infusion route, cell dose, follow-up period, design of the study (i.e., randomized, blind/double bind, and placebo controlled), statistical power, and clinical end point(s). Such sequence has been followed in a number of clinical protocols where MSC-based products have been used to treat patients with cardiovascular diseases. In these cases, assessment has included (1) infusion route (intracoronary, trans-endocardial, intramyocardial, intravenous, and intramuscular), (2) cell dosage, (3) proper timing between manufacturing of the cell product (availability), (4) time of infusion in relation to progressive changes after disease onset (i.e., remodeling after MI), and (5) the diversity of techniques to assess improvement (i.e., cardiac perfusion and leg revascularization) [31,77,98,101,111,133]. Despite the above complexity, efficacy assessments have yielded promising results that have unveiled that MSC-based therapies may bring significant improvement to the quality of life of patients suffering from various types of diseases [16,38].
However, MSC-based cell therapy, a relatively new component in the arsenal of therapeutic options for several medical condition diseases, still has a rather long way to go in order to become not only a competent, eligible, and widely accepted treatment, but also devoid of carrying risks of malignancies and/or other serious complications.
Results of a number of cellular and animal studies have posed the question whether after infusion, migration and homing of MSCs to a particular target tissue may generate a cellular and molecular microenvironment favorable to the growth and metastasis of “resident” tumor cells [134,135].
Similarly, the described differentiation-related transition of MSCs from an immunoprivileged to an immunogenic condition [81] may seriously affect the outcome of a clinical trial using allogeneic MSCs by exposing the receptor not only to the rejection of the graft but also to other contrasting immunological.
Many safety questions remain with respect to the therapeutic use of MSCs. The answers to which will vary depending many factors, including the disorder to be treated, the source (bone marrow, cord blood, and fat), origin (autologous or allogeneic), and manufacturing of the cell product. Additionally, other changeable conditions (cell dose, infusion route, and follow-up period) may influence the safety outcome of a particular MSC-based therapy. Whereas the potential deleterious capability associated to use of MSCs is a concern, most studies so far do not support this notion [136,137].
Regulations for the Clinical Use of MSCs
The formulation of a cell therapy protocol aimed at repairing damaged or diseased tissues and organs has been in most cases sustained by the translation of molecular, cellular, and/or animal data into a clinical setting. However, in addition to the enduring significance of the preclinical information, it is also important to consider that any proposal for utilization of MSCs in a clinical study should be in agreement with policies imposed by the respective national regulatory agency. These agencies should regulate patient selection, the sequence of processes involved in product manufacturing and testing, infusion procedures, evaluation and follow-up of preliminary and persistent signs of efficacy, and/or side effects. This appears to be the case with biomedical regulatory agencies in several countries, among them the United States, the European Community, Japan, and Canada and to some extent, India, China, and Brazil. On the other hand, in the vast majority of countries, regulations and/or legal restraint that rule the clinical use of adult stem cells are either under development or completely nonexistent (
Regulations for the manufacture and use of MSC-based cell products are focused, among other basics, on the following requisites: minimal manipulation procedures during manufacturing, no combination with another article, and intended for autologous or allogeneic use in a first- or second-degree relative. In addition, the cell product must be designed for homologous use, which in the case of an MSC-based cell product means utilization merely for the repair or replacement of a distinctive (homologous) mesoderm-derived tissue (bone, cartilage, muscle, marrow stroma, and connective tissues from dermis and nerves). The last requisite does not include other possible “homologous” tissues [138–139]. If an MSC product complies with the above requisites, it will be regulated solely under the U.S. Food and Drug Administration-Public Health Service Act (FDA-PHS) Section 361/21 CFR Part 1271. Accordingly, requirements for marketing options include, among others, solely premarket reviews, inspections, and enforcement approval. If the manufacture and intention of use of an MSC product do not fulfill the above requisites, the product ought to be regulated under FDA-PHS Section 351. Consequently, the marketing options require an Investigational New Drug (IND) Application (clinical trial), a Biologics License Application, and a strong evidence that the cell product is safe, pure, and potent. In addition, the inevitability to acknowledge the permanent, softly meticulous, and bureaucratic barriers established by the FDA.
To date (at least in the United States) there are no MSC products that meet the terms of 361 regulations. Consequently, the clinical use of an MSC-based cell product will be governed by the regulatory policies of a clinical trial. It is noteworthy to emphasize that only after full completion of the three required phases of a clinical trial, the manufactured cell product can be marketed and thus used to treat patients. In terms of real time, the use of an MSC-based product for treatment of a particular disease will take at least 6 years, after IND approval and initiation of phase I studies (which easily can take another year). This long waiting time is extremely significant for patients suffering from one of the so-called “rare” diseases (at present without therapy). For these patients the design of a stem-cell-based protocol and its clinical initiation encloses a silent, but noteworthy, message. Despite these patients having no illusion of a panacea, the initiation of a clinical trial symbolizes the long-time awaited right of hope. Often the expectation of these and other patients has been shattered by an excess of sophisticated, bureaucratic, and sometimes unnecessary guidelines imposed by the regulatory agencies. Even in 2001, when guidelines for the clinical use of stem-cell-based products existed (theoretically) with less paraphernalia, regulations occurred to be as fiercely as today. Probably, this was the reason why a group of biomedical researches [112] started in the United States a stem-cell-based clinical study for Amyotrophic Lateral Sclerosis patients without the venia of the FDA. Promisingly, the attempt of the European Commission to launch the Cascade Program (EU, HEALTH-F5-2009-223236), as well as other regulated programs in other regions of the world, stands for a realistic and urgent necessity to develop clinical studies, having as the predominant aim in mind the welfare of the individual patients in such clinical trials.
Footnotes
Acknowledgments
TCA Cellular Therapy, LLC, is a private research company owned by health care professionals. The funding to perform the research has come from individual physician investors who strongly believe in stem cell therapies.
We acknowledge Dr. Vicki Nicely, Janet Jones, and Gerhard Bauer for critical review of the article.
Author Disclosure Statement
All authors indicate no potential conflicts of interest.