Insights into bone marrow‐derived mesenchymal stem cells safety for cutaneous repair and regeneration

Abstract

Wound healing involves the orchestration of a complex process of interactions between numerous types of cell, components of extracellular matrix and signalling molecules following injury, which is usually a highly successful biological course to reconstruct the integrity of the skin. Nevertheless, when skin is severely damaged, the injured skin is limited in its ability to repair itself and possibly results in the hypertrophic scars or so‐called keloids, and non healing wound or ulcer. Bone marrow‐derived mesenchymal stem cells (BM‐MSCs) are being clinically explored as a promising therapy in the field of tissue repair and regeneration. However, potential risks associated with these cell‐based therapies remain uncertain. The aim of this review is to summarise the safety issues accompanying the administration of BM‐MSCs for acute or chronic skin repair and regeneration. More importantly, this review highlights the requirement for fundamental research to improve future clinical application of these strategies, as well as for regulatory authorities to establish clinical criteria to identify the qualitative requirements for the manufacture process of cells products, which will ensure the manufacture process of the best benefit‐to‐risk ratio of cell‐based therapy for the patients.

INTRODUCTION

In recent years, mesenchymal stem cells (MSCs) have captured considerable interests in improving cutaneous repair and regeneration. MSCs are powerful multipotent progenitor cells, which can be readily isolated, expanded and transplanted, especially MSCs derived from bone marrow (BM), and their application in cell‐based therapy has become a major focus of research. Since the first clinical trials carried out with adult stem/progenitor cells offer new hope for patients (1), increasing investigators have initiated the characteristic exploitation of cells in numerous animal models or preclinical trials. However, the cells cannot be used widely in clinical treatment. This situation may be explained, at least in part, by potential side effects affecting the safety profile of these cells not only in a short‐time period following administration but in potential long‐term risks associated with MSCs therapy. For example, MSCs administrated into acute glomerulonephritis in rats exhibited the beneficial effect, but intraglomerular MSC can maldifferentiate into adipocytes that obstructed the normal function of the kidney and lead to chronic kidney disease (2). At present, there are yet few concerns the safety of MSCs therapies in the field of cutaneous repair. From a regulatory perspective, these advanced therapeutic strategies must be ensured with undoubted safety and effectiveness not only in successful isolation and efficient culture but also in clinical administration of products of viable cells. We hope the issues discussed in this review would arouse the investigators to ponder the safety when using these cells for tissue repair.

MSCs APPLICATION IN CUTANEOUS REPAIR AND REGENERATION

Skin is a complex tissue (3) and the largest organ of the body, which is mainly responsible for the protection of the body, sweating, feeling the hot, cold and pressure, and thus a ‘full‐thickness' wound would result in damage to many structures as well as cell layers and lineages. When skin tissue is damaged, the process of wound repair takes place in a regulated mode under ideal situation, which plays a pivotal role for tissue homeostasis. However, tissue repair becomes seriously dysregulated in many cases and contributes to numerous pathological problems, such as scars, chronic wounds, and so on, which remains a challenging issue in clinic. With the expansion of cutaneous physiology and pathology research and the rapidly development of stem cell biology, the application of stem cell technology brings hope to solve the matter following injury and provides a new method for skin regenerative medicine research. In recent years, animal studies and preclinical trials have demonstrated that bone marrow‐derived mesenchymal stem cells (BM‐MSCs) extracted from autologous or allogenic BM may play an important role in skin wound healing due to their huge potential therapeutic value.

As stromal cells in the BM, MSCs have been known to contribute to the cutaneous homeostasis and wound healing. And, as with many cell‐based therapies, MSCs act through complex interactions with the endogenous cells and tissues, and the mechanisms of the MSCs on wound repair distinguished from different investigators (Figure 1). Several recent reports have implied that MSCs possess the capacity to differentiate into skin cells and lineages 4, 5. When cultured using epidermal growth factor, keratinocyte growth factor, hepatocyte growth factor (HGF) and insulin‐like growth factor‐II (IGF‐II), human BM‐MSCs may differentiate into epithelial‐like cells by characterising the surface phenotypes of these cells, involving cytokeratin 19 and cytokeratin 18 (4). Culturing MSCs with growth factors may cause phenotypic changes of the cells, which indicated that MSCs may directly differentiate skin epithelial cell in vitro. In the other hand, another group reported that MSCs can contribute to trauma repair via processes involving MSCs differentiation various skin cells in vivo study (5). Mikako Sasaki and colleagues injected MSCs derived from green fluorescent protein (GFP) transgenic mice into the sites of mouse wounds, and then ascertained GFP⁺ cells associated with the specific markers of skin cells type including keratinocytes, endothelial cells and pericytes (5). However, the view is controversial for differentiation of MSCs to epithelial cells, and many investigators believed that the efficacy of MSCs is limited in the aspect. Some researchers have distinct opinion suggesting that MSCs predominantly promote re‐epithelisation of skin, vasculogenesis, collagen deposition through secreting a number of soluble factors related to wound healing 6, 7, 8. Chen et al. have shown that BM‐MSCs could secrete the growth factors and chemokines recruiting macrophages and endothelial lineage cells, and concentrated BM‐MSC‐conditioned medium applied to a mouse model of excisional wounds could accelerate wound healing (8). In apparent agreement with this, a study in mice in vivo reported that MSCs improved limb function and appearance by increasing levels of basic fibroblast growth factor and vascular endothelial growth factor (VEGF) protein (6). Furthermore, other researchers 9, 10 also came to a similar conclusion in clinical study that local administration of MSC can improve the radiation burn healing process through paracrine factor production including cytokines and trophic factors that may make the absence of clinical symptom of radiation inflammatory waves. The findings also demonstrated that there was safety and no side effect after MSCs autologous administration. Therefore, it is obvious that the paracrine or autocrine factors of BM‐MSCs appear to play a central role for the heightening trauma healing with respect to cutaneous repair by releasing high levels of angiogenic, antiapoptotic and mitogenic cytokines such as VEGF, HGF, angiopoietin‐1, adrenomedullin, platelet‐derived growth factor‐BB 8, 11 and IGF‐I 12, 13, 14. It has been referred that MSCs work as trophic mediators in wound healing (15). In addition, some people believed that MSCs can influence on re‐epithelisation of skin. For example, a preclinical study in 2008 confirmed the truth of MSCs contributing to allogeneic skin graft transplantations and inducing re‐epithelisation in the epidermis (16). Moreover, MSCs are also able to contribute to the skin lesion by collagen deposition rather than differentiation. Some investigators have also observed the MSCs derived from BM can promote cutaneous wound repair by mainly contracting a collagen matrix and transcribing both collagen types I and III after injury (17). Besides mechanical injury, the efficacy of MSCs in pathological wound, for instance epidermolysis bullosa, was also studied in clinic. Conget and coworkers reported that allogeneic MSCs by intradermal administration can promote re‐epithelialisation of chronic ulcerated skin and type VII collagen replenishment (18). Another characteristic of MSCs appears to have potent anti‐infammatory effects resulting from injury. Several studies in murine models of injury and disease have documented mouse (19) and human (20). MSCs engrafted to regions of inflammation and tissue damage. Murine syngeneic and allogeneic MSCs applied to excisional wounds in mice demonstrated an equivalent efficient to attenuate the local inflammatory response and enhance wound healing (19). Additionally immune properties of MSCs cannot be ignored. As early as in 2002, Baboon MSCs have been observed to suppress lymphocyte reactivity to allogeneic cells, which prolonged skin graft survival and accelerated the process of the wound healing (21). It may be certify that the immunoregulatory traits of these cells may work in future applications of wound repair and regeneration 8, 21. Recently, there are also some groups to concern about the regeneration of appendages including hair follicles, sweat glands and sebaceous glands. In one study, CM‐DiI fluorescence‐labeled Flk‐1⁺ BM‐MSCs derived from BALB/c mice (white hair) were infused into lethally irradiated C57BL/6 mice (black hair). The recipient mice grew white hairs about 40 days later. Immunochemistry staining and reverse transcription polymerase chain reaction indicated that skin tissue within the white hair regions contained cells derived from donor MSCs (22), implying that BM‐MSCs are significantly involved in the regeneration of functional hair follicles of skin tissue. Consistent with the above observation, in another study, using a similar mouse model, GFP⁺ BM‐derived cells injected around the wound and applied to the wound bed expressed the keratinocyte‐specific protein keratin and formed glandular structures, suggesting that BM‐MSCs promoted wound healing and the regeneration of skin appendages (14). The previous study of our laboratory preliminarily demonstrated that MSCs affected the regeneration skin appendages reconstruction after trauma, especially sweat glands (23). Moreover, the improvement of scar after trauma has been a growing attention. When skin is severely damaged, the injured skin is limited in its ability to repair itself and possibly results in the hypertrophic scars or so‐called keloids, which implicates an undesirable consequence in appearance and function of regeneration (24). In a preclinical study, the results have demonstrated the efficacy of MSCs in preventing contraction of split‐skin grafts (25). When skin grafted, the area of the treatment of BM‐MSCs has less contracture, less scarring, better elasticity and more smoother than the area at the control site during a 2‐year follow‐up analysis. Similar to the situation, the chronic non healing wounds are not well treated, which will lead to immune dysfunction and increase the chances of infection. BM‐MSCs recently appeared to contribute to refractory healing by multiple mechanisms that could result in better wound healing. Vojtassák et al. have demonstrated that the cells can work in decreasing wound size and increasing the vascularity of the dermis (26), which suggested that BM‐MSCs may aid the healing of chronic lower extremity wounds in clinical cases. These issues have broad global implications (27). Taken together, these findings have notably improved our understanding to the contribution of BM‐MSCs in cutaneous physiology and pathology, and opened a new avenue to study the mechanisms of cutaneous homeostasis and regenerative healing.

Figure 1.

The natural functions of the mesenchymal stem cells (MSCs) on cutaneous wound repair. AM, adrenomedullin; Ang‐1, angiopoietin‐1; HGF, hepatocyte growth factor; IGF‐I, insulin‐like growth factor‐I; PDGF‐BB, platelet‐derived growth factor‐BB; VEGF, vascular endothelial growth factor.

Although the results are encouraging, it is almost preclinical or animal experiments, and there are currently only a few large‐scale clinical studies. One of the most important reasons is that the problem of safety has not been resolved. The aim of this review is to present an overview of MSCs therapy confronting with a variety of problems, with a particular focus on the issue of the safety of MSCs administration. Since BM‐MSCs are the main target cell population for the cell therapy, one important issue is to investigate the safety of the cell‐based the application. Accordingly, the process of cell preparation has a number of different points to consider compared to guidelines developed for finished drugs. Studying of stem cell security also offers the particular opportunity to define fundamental principles during the process of the clinical implementation.

SAFETY

MSCs engraftment is one of the most cutting‐edge treatments referring to MSCs own collection, and then being cultured, purified and back to input into the body for the patients with wound, scleroderma, dermatomyositis, lupus erythematosus, rheumatoid, motor neuron diseases, and so forth cutaneous diseases. If the cells are applied in clinical therapeutics, how ensures the delivery of a ‘cell drug’ that is safe, reproducible and efficient? Are cell separation, culture, translation, identity and the numbers of engraftment controlled by stable evolutionary rules? Currently, guidelines for clinical grade MSC production are now proposed in Europe and various quality controls are now systematically performed among which some are considered as released criteria such as the sterility, the cell viability and the karyotype. The process of cell‐based therapies is considerably complex, so a great many of fundamental biological questions remain to be answered. In fact, the application of human MSCs could be confronted with many safety issues as well as other cell‐based therapies, but it could also face the peculiar problem of stem cell, which will be discussed reasonably in the following paragraphs.

BM‐MSCs separation, culture and purification

BM‐MSCs compared with other organisations were relatively larger, but it is difficult to meet the needs of cell therapy because of the number of only 0·001–0·01% accounting for nucleated cells derived from BM (28). For clinical purposes, it is necessary to produce large amounts of cells in vitro to suffice the requirements. Human MSCs isolated from BM can be readily expanded by multiple passages in medium containing fetal bovine serum (FBS) (29), because of the physical property of their adherence to the tissue culture plastic 28, 30. However, it may be appear such several issues in the whole progress involving cellular separation, proliferation and survival. When the cells are harvested, it is necessary for patients to assess the overall situation before extracting BM‐MSCs. The operation will harm the health of the sufferers with the contraindications such as infection, renal crisis, pericardial effusion, and so forth. And in terms of the cells product itself, ensuring sterility is the primary concern for tissue‐based therapy. Since viable cell‐based products cannot undergo a terminal sterilisation step, as is the same case with other pharmaceuticals, they need to be produced under sterile conditions (31). So, all parenteral drugs and medical devices must be high‐temperature sterilisation, and disposable items disinfected and microbiological testing of aerobic and anaerobic bacteria and fungi before application can guarantee the cells in a sterile and not bring harm to patients. In addition, all cell culture conditions are extremely important in maintaining the properties of large numbers of viable cells (32). Currently, the medium used for cells culture in general would be joined 10–20% the FBS or fetal calf serum (FCS). Although FBS or FCS seems to be essential for obtaining high‐quantity and quality MSCs (33), it can bring to potentially transfer disease, or even cause immunogenic reactions in the patient 34, 35, including transferring immunogenic xenoproteins and infectious agents, especially transmissible spongiform encephalopathy (36), which compromise the clinical effectiveness of MSCs transplants (37). Therefore, it is very necessary to try to reduce any potential risks for patient by preventing the use of non human products in culture process (38), and the substitution of FBS/FCS or serum‐free medium is being examined. In one report, it has been attested that platelet lysates were more efficient for ex vivo expansion of MSCs than FCS and can maintain the capacity of MSCs differentiation (39). Moreover, serum‐free medium improved has applied the culture of the cells in our lab, and the outcome indicated that the proliferative abilities of BM‐MSCs with serum medium were almost similar to that of BM‐MSCs with serum‐free medium improved.

Additionally it cannot be avoidable for the external components pollution in the culture course of events. Isolation, purification, amplification and introduction of the long preparation need to add in antibiotics, growth factors, antibodies, collagenase, protease, serum and other materials before a cells product can be released. Other exogenous factors including bacteria, mycoplasma, viruses, and so forth also affect application of the cells. Of course, for the viruses, the main source is the blood of the donor. The serologic tests of the donors are necessary in order to analyse for the presence of human immunodeficiency virus, hepatitis B and C viruses and human T‐lymphotropic virus (40). Another very important issue to note, many viruses have long latency period, which means that the potential risks in the training process might be unable to be eliminated completely.

In vitro operation, expansion, proliferation, differentiation, the properties of the cell may gradually change and bring the transformation of the biological function of stem cells, such as telomerase activity, karyotype, histocompatibility antigens, the original tumor suppressor gene, proliferation, differentiation potential and other changes to bring the immune toxicity and oncogenicity risk. For this reason, it is not advisable to be maintained for long periods in culture, and they may begin to ‘drift’ or acquire genetic and epigenetic changes (41). In addition, there is often a need to perform extensive over large periods in vitro at the time of culture (42), preservation, transportation, recovery, preparation, which is difficult to guarantee the cell survival rate, biological functions and uniform stability of BM‐MSCs. Even though at early passages MSCs can experience chromosomal abnormalities (43), which indicates that the properties of MSCs should be monitored to warrant the security of MSCs expanded in vitro used for cell therapy.

BM‐MSCs storage

These cell products are likely to have a short shelf‐life (44), which often mean that these products should have been administered to patients before the potency of the cells has changed. In general, the final cell products are cryopreserved in cell bank before delivery to the patient. It is necessary to consider both the product consistency and the cell stability. For the product consistency, stem cells can be affected by different donors including age, sex, physical condition, and so forth, which may be different in growth rates, differentiation capacity, karyotypic stability, the ability to infusion and activity in vivo (45). Taking the age into consideration, MSCs' ageing could happen with donors' age and the biologically active of cells seems to get worse. Each lot of the cells will be specific to one patient for the individualised therapies taking into account human leukocyte antigen (also known as transplantation antigens) matching. It has shown that the rejection is the biggest obstacle of allogeneic transplantation success in clinical practice. Besides, the cells' stability is also an issue to be paid attention during the storage. The cells' product is different from other conventional drugs, because it is ‘live’ substance with the limitation of source and differences in the production process and storage conditions. So, it is very important to test the stability profile of therapeutic products before implantation, including expression of key genes, genomic stability and epigenetic stability (methylation, miRNA, histone acetylation and X‐chromosome inactivation).

BM‐MSCs administration

There are two methods in common using for the transplantation ways in tissue healing: (1) local administration: to directly target the skin lesions by single or multiple injection sites. (2) Intravenous administration: MSCs amplified infusing through the bloodstream to the lesion. However, it has not yet been definitely defined for the dose of MSCs that is suitable effective, and in contrast not harmful, for various clinical indications (46), which needs to continue to explore. Besides these, there are other several points to consider including potential maldifferentiation, immunosuppression and instigation of malignant tumor growth after implantation (47).

BM‐MSCs grafted directly within the skin may produce local pathology of yet unknown consequences and intravascularly administered MSCs largely do not enter the lesions but rather lodge predominantly organs 48, 49, such as lung, heart, spleen, kidney, liver, which implies that it would cause different influence to the body in the light of the site of injection, especially intravenous administration. When systemic administration of cells contaminated bacteria, mycoplasma, viruses brings the tremendous risks of acute toxicity, abnormal toxicity and pathogen infection to patients, and severe infection with the blood into the body may be lead to life threatening. Endotoxin or pyrogenicity testing is requirement of all cell‐based therapies. Patients caused acute death may be due to vascular obstruction, pyrogen reactions, acute infections, sepsis, acute toxicity, complications, bleeding, allergic, and so forth by poor stem cell products. In addition, low immunogenic potential of BM‐MSCs together with their effects on immune response make them a promising method of treatment for severe refractory autoimmune diseases, but knowledge regarding the body's immune response to cell‐based products will also influence the safety of the product. Systemic administration of MSCs may escape immune surveillance, which makes the recipient more susceptible to opportunistic infections. When transplanted with cancer cells, MSCs can adapt a tumor‐associated fibroblast phenotype and support the growth of the cancer by directly or indirectly promoting tumor growth, metastasis and angiogenesis. There have been some animal studies to reveal that MSCs transplanted enhanced tumor growth when tumor cells were implanted 45, 50. It is unclear whether the MSCs promoted tumor growth by immunomodulatory, trophic or other effects. Moreover, of note, it is important to remember that once these products are recognised by the host as “foreign”, the risk of rejection and associated pathologies may be greater than the potential benefit to the patient (51). Indeed, systemic administration of MSCs was found to take effect for the treatment of experimental models of diabetes (52), rheumatoid arthritis (53), systemic lupus erythematosus (54) and multiple sclerosis (55). BM‐MSCs cannot be made from the patient himself, and the cells used turn to donors, which means that patient's immune system has the possibility to fight them as foreign intruders.

BM‐MSCs are in a place where they do not belong to, which may be present some certain the risks. It has the mounting recognition that cells with ‘stem‐like’ properties reside in nearly all kinds of tissues and generally resemble each other, such as nervous system, BM, peripheral blood, fat tissue, skin, gut, and so forth. Along with the explosive growth of stem cell field over the past few decades, it was quite a revelation that the cells with ‘stem‐like’ had been regarded as deterministic and rigid – beyond embryogenesis adulthood – with the potential for assuming multiple fates in response to variable environmental and regional cues (56). MSCs derived from the stroma of BM are administrated into cutaneous lesions, which leave the original microenvironment/niche and enter into the new ‘home’. If MSCs going in a new niche will differentiate into the cells expected, and the cells with ‘stem‐like’ properties may not generate mass, however, there may be the pluripotency of cells allowing generation of a wide array of differentiated cell products and entailing the possibility of teratoma formation and the presence of unwanted cell types (41), even where the cells may reside indefinitely. Of course, MSCs could be tumorigenic through other patterns. For example, when Wnt signalling is suppressed, MSCs have the potential to transform into sarcomas (57). It may not be surprising that a cell type not from skin lineage will proceed its natural tendencies under the most stressful and pathological conditions. So it should be avoid the phenomenon in the field of tissue repair and regenerative medicine. Unfortunately, it is under the most severe case of a disease that has to call upon cell transplantation as a therapeutic option.

CONCLUSION

As shown in this review, there are many issues to consider when preparing a cell‐based clinical therapy. The routings carried out including acquisition, procurement, database, preparation, preservation, testing, evaluation, transmission, transfusion are non standard. Furthermore, understanding of human stem cells is still immature stage. It is unclear for mechanism of action, fate of the cells in vivo, long‐term implantation effects and how to terminate adverse reactions after transplantation. So, it is not doubt that stem cells abusing will bring disastrous consequences, and especially in developing countries, the phenomenon is common. It is obvious that there is currently a significant lack of standardisation in MSCs manufacturing protocols as well as in defining release criteria and potency assays. In the view of these, regulatory bodies expected will establish a set of more consummate global standards and guidelines for cell‐based therapeutics. Of course, as the US Food and Drug Administration's Good Tissue Practices Final Rule have established a tiered system based on the level of risk associated with the cell product, which can regulate the cell therapies in late‐stage clinical trials. It can be anticipated that the safety of MSCs can be strictly supervised and MSCs will be gradually introduced into clinical practice for a number of serious diseases within an even shorter period of time after successful conclusion of ongoing preclinical and clinical tests.

ACKNOWLEDGEMENTS

This study was supported by the National Basic Science and Development Program (973 Program 2012CB518105), the Postdoctoral Science Foundation (20080440225) and the National Natural Science Foundation of China (81121004, 81000843).