Umbilical cord-derived mesenchymal stem cells: strategies, challenges, and potential for cutaneous regeneration

Abstract

Umbilical cord mesenchymal stem cells (MSCs) are a unique, accessible, and non-controversial source of early stem cells that can be readily manipulated. As the most common pluripotent cell, bone marrow-derived MSCs display limitations with the progress of stem cell therapy. By contrast, umbilical cord-derived cells, which have plentiful resources, are more accessible. However, several uncertain aspects, such as the effect of donor selection or culture conditions, long-term therapeutic effects, product consistency, and potential tumorigenicity, are the bottleneck in this clinical therapy. MSCs are predicted to undergo an unprecedented development in clinical treatment when a generally acknowledged criterion emerges. In the current paper, we highlight the application of umbilical cord-derived MSCs in skin therapies based on our previous studies, as well as the achievements of our peers in this field. This paper focuses on the strategies, challenges, and potential of this novel therapy.

Keywords

umbilical cord / mesenchymal stem cells / cutaneous regeneration

Introduction

The wound healing process, particularly in the skin, has been extensively studied and reported in a considerable number of documents. Several novel therapies for skin repair have emerged, extending back to more than 100 years. Among the many treatments, autologous skin transplantation remains the clinical “golden standard.” However, because of the shortage of skin resources and the inevitability of new wounds, autologous skin transplantation has limitations in treating large-area skin damage. Thus, cell-based therapies are attractive candidates in regenerative medicine to treat many conditions in which present conventional treatments are inadequate. Currently, both scientists and researchers are focusing on umbilical cord-derived mesenchymal stem cells (WJMSCs) to improve wound healing after a severe injury. However, a significant gap between the promising laboratory-based research and approved final therapies in this emerging field remains. Thus, the current review aims to outline the three main aspects, namely, the strategies, challenges, and potential, of WJMSC application in cutaneous regeneration.

Umbilical cord MSCs (UCMSCs): potential role in regenerative medicine

The umbilical cord represents the link between the pregnant woman and the fetus during gravidity. It is composed of a special embryonic mucous connective tissue called Wharton’s jelly, which lies between the umbilical vessels and the covering amniotic epithelium [1]. The human umbilical cord has an average weight of 40 g, and its length reaches approximately 60 cm to 65 cm. It has an average diameter of 1.5 cm at term [2,3]. It is covered by a single/multiple layer(s) of squamous-cubic epithelial cells [4,5] called the umbilical epithelium, which is commonly thought derived from the amniotic epithelium. These epithelial cells exhibit ultrastructural and functional characteristics similar to those seen in keratinocytes [6] and were shown to possess a stem cell nature [7]. Increasing evidence suggests that human umbilical cord-derived cells share a considerable amount of common properties with MSCs. Thus, their differentiation potency in conditioned cultures specifically designed to differentiate MSCs and other adult stem cells from certain lineages must be investigated.

MSCs in the umbilical cord can be isolated from umbilical cord blood, umbilical vein subendothelium, and Wharton’s jelly (Fig. 1). Whether MSCs isolated from the different cord compartments represent different populations remains to be determined. Nevertheless, the Wharton’s jelly-derived cells (WJCs) sharing the same surface marker with the MSCs indicate that they are affiliated to the MSCs. Wharton’s jelly-derived MSCs (WJMSCs) are derived from the cushioning matrix between the umbilical blood vessels rather than from umbilical blood [8]. The cluster of differentiation 73 (CD73), CD90, CD105 and human leukocyte antigen (HLA) class I are positive, whereas CD45, CD 34 and HLA class II are negative. In the present study, UCMSCs are referred to as WJMSCs.

These WJMSCs have several common properties, such as poor capability to differentiate into adipocytes [9-11], shorter doubling times compared to bone marrow-derived MSCs (BMMSCs), and greater number of passages to senescence [9-13]. The umbilical cord blood-derived MSCs (UCB-MSCs) may express granulocyte-macrophage colony-stimulating factor (GM-CSF) similar to WJMSCs, although this finding has not been constantly observed [14,15]. A fast doubling time is a common feature of MSCs derived from fetal blood [16], cord blood, and Wharton’s jelly. The test of colony forming units (CFU)-F suggests that cells derived from Wharton’s jelly have a higher forming frequency [9]. This shared feature possibly reflects the relatively primitive nature of these MSCs compared to BMMSCs.

WJMSCs are plastic-adherent, multipotent, and robust; these characteristics can also be observed after freezing and thawing. The capacity to be engineered to express exogenous proteins demonstrates that WJMSCs meet the basic criteria used to define adult-derived MSCs. WJMSCs exhibit remarkably faster proliferation and greater in vivo expansion ability than BMMSCs, which may be due to the induction of telomerase expression [8]. Similar to BMMSCs, WJMSCs express the stem cell factor gene [17]. To date, human WJMSCs have been proven to induce the formation of various tissues such as bone, cartilage, and adipose cells [18-21]. They have been successfully confirmed to differentiate into endothelial cells [22] after the addition of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF) to cultures, which are critical to cutaneous repair. A comparison of the different MSCs is summarized in Table 1.

WJMSCs are non-immunogenic upon first injection into allogeneic recipients. However, their repeated injection elicits an immunogenic response. Moreover, WJMSCs are immunogenic when they are exposed to interferon before injection or when they are injected into inflamed skin. The current essay confirms the immunogenicity of WJMSCs in vivo and its allogeneic use in disease tissues.

WJMSC application in regenerative medicine

The differentiation potency of WJMSCs in conditioned cultures, such as the bone [23,24], skin [25], endothelium [26], hepatocyte [27], and neural lineages [28], had been confirmed. Therefore, WJMSCs have tremendous potential as a stem cell source in regenerative medicine applications.

Compared with BMMSCs, WJMSCs can form premature adipocytes with smaller multilocular lipid droplets [9]. Both type II and type I collagen fibers are detected [9,29] by immunohistochemistry [9] or by hydroxyproline assays [29]. Osteogenic potency was first demonstrated in 2004 [20] in the formation of alkaline phosphatase-positive aggregates and nodules accompanying the expression of osteopontin, which is an osteospecific matricellular protein. Recently, WJMSC differentiation into skeletal myocytes has been successfully confirmed both in vitro and in vivo [21]. Using a medicinal herb, some researchers induced human WJMSCs to differentiate into neuronal lineages [30] and found that WJMSCs, when treated with the herb extract, express pleiotrophin. Pleiotrophin is a secreted growth factor that induces neurite growth and promotes mitosis for epithelial, fibroblast, and endothelial cells [31]. In addition to pleiotrophin, FGF-2 and brain-derived neurotrophic factor were also found secreted by WJMSCs [13].

WJMSCs have recently received considerable interest for application in clinical treatments because of the preclinical trial results of their potent capability to treat many devastating diseases in animals. MSCs have also been shown to act on several levels of endogenous repair, resulting in disease resolution, cell protection from injury, and direct boosting of tissue repair [32,33]. When administered to animals with acute renal failure, MSCs prevent apoptosis and induce the proliferation of renal-tubule epithelial cells in a differentiation-independent manner [34,35]. When injected into the subsequent myocardial infarction, MSCs can reduce the formation of scars [36-38]. When administered to prevent the onset of insulin-dependent diabetes mellitus, MSCs can protect the β-islets from autoimmune attacks. When administered after the onset of the disease, they promote a temporary restoration of glucose regulation, indicating protection and repair of damaged islet tissues [39]. Apart from directly boosting tissue repair, UCMSCs have also been shown to regulate the immune system and reduce the tissue damage caused by excessive inflammation.

MSCs have been shown to directly affect the innate and adaptive arms of the immune system [40], promote the neovascularization of ischemic tissues [41], and enhance the proliferation of epithelial cells [42]. The immunomodulatory properties of WJMSCs are similar to those of BMMSCs [43]. MSCs appear to have the unusual capability of evading the immune system. As shown in initial clinical trials, both autologous and allogeneic MSCs can be transplanted without immune rejection [44,45]. Further preclinical studies showed that human MSCs can engraft and be maintained in many fetal and adult sheep tissues without apparent rejection [46]. MSC injection in baboons can prolong the life of a transplanted skin graft and suppress T cell proliferation in a dose-dependent manner [47]. In conclusion, MSCs may drive or allow dendritic cells (DCs) activity, which serve as the major link between innate and adaptive immunity because of their phenotype, to further weaken T cell-mediated immunity.

UCMSCs are also more efficient in terms of stem cell potency compared to BMMSCs. In addition, the shorter doubling time of cultured WJMSCs results in an easier and rapid propagation compared with that of BMMSCs. In addition, feeder layers or high serum concentrations are not necessary for WJMSC expansion. With the distinct advantages of WJMSCs, such as accessibility with little or no ethical concerns, as well as painless procedures to donors with lower risks of viral contamination, WJMSCs should be considered as an alternative to BMMSCs.

WJMSC-based strategies in skin repair

The skin is a complex tissue and is the body’s barrier to the outside world [48]. Hence, severe skin trauma or burns will lead to a dysfunction of the entire body. The level of skin repair directly affects the living quality of patients. MSCs have been shown to possess a strong ability to improve tissue damage in response to skin injury by contributing to collagen deposition [49], wound contraction [50], angiogenesis [51], regeneration of skin appendages, and enhanced growth of epidermal cells [52]. Currently, most of the studies related to MSCs in clinical treatments are on BMMSCs; only a few studies mentioned the application of WJMSCs. The two sources of MSCs, namely, the umbilical cord and the bone marrow, have similar characteristics. However, the process of obtaining the umbilical cord-derived MSCs is much easier and does not harm the donor compared with that of BMMSCs. Given the distinct advantage of WJMSCs in regenerative medicine, they are considered an alternative source of stem cell-based tissue engineering, which is a promising strategy for the restoration of injured skin.

BMMSCs can adapt to the culture conditions of the skin, but do not differentiate into epithelial cells. Carlin [53] repeated these experiments with WJMSCs because of their advantages in regenerative medicine. Similar to BMMSCs, MSCs derived from Wharton’s jelly can adapt to the culture conditions of the dermal equivalents (DEs), suggesting that WJMSCs confer a therapeutic benefit by supporting the regeneration of the dermal compartment [25]. Interestingly, a small population of WJMSCs coexpresses the mesenchymal marker vimentin and the epithelial marker pan-cytokeratin (CK). By contrast, BMMSCs are CK-negative after isolation and on DEs. Compared with BMMSCs, WJMSCs can survive on DEs and can more easily adapt to the culture conditions of the skin. Therefore, WJMSCs can promote skin epithelization, vasculogenesis, and collagen deposition by secreting a number of soluble factors during the wound-healing phase. Thus, WJMSCs can be used as an interesting and promising tool to regenerate skin wounds. The restoration of cutaneous appendages after a severe skin injury, which is related to the function of the regenerated skin and affects the quality of life, may be an important function of WJMSCs during skin repair.

The regeneration of sweat glands after deep burns has been an unresolved challenge. To address this problem, our group previously induced BMMSCs to acquire the phenotype of sweat gland cells in vitro and then transplanted them into fresh skin wounds made by excising the anhydrotic scars of five patients after their deep burn injuries were healed [54]. The MSCs transformed into sweat gland cells and facilitated the recovery of functional sweat glands. This phenomenon may help address the problem of sweat gland depletion in patients surviving extensive deep burns [54]. WJMSCs in a specific induction system may hopefully differentiate into sweat gland cell-like cells. Recently, WJMSCs were successfully induced to differentiate into sweat gland cells in vitro [55]. Findings from our study [55] indicate that WJMSCs can differentiate into sweat gland-like cells via a novel and feasible system that is more effective than our previous method [54]. WJMSCs, as a novel source of stem cells, can differentiate into sweat glands for further regeneration of the epidermis and skin appendages.

The umbilical cord is a rich source of progenitor cells, including MSCs and endothelial colony-forming progenitor cells (ECFCs), with high proliferative potential. Both cell types play key roles in maintaining the integrity of tissues and are probably involved in regenerative processes and tumor formation. However, clinical-grade human MSCs have been expanded in vitro for tissue engineering or immunoregulatory purposes without standardized culture conditions or release criteria. Given that cellular therapeutics must eventually find their way from the bench to the bedside, a new method of obtaining progenitor cells without the use of animal proteins is important for clinical treatment; that is, to completely avoid cell contact with xenogeneic proteins. Replacing fetal bovine serum with pooled human platelet lysate (PHPL) in all steps of currently existing protocols or the use of serum-free medium is one approach to addressing this issue.

The essay by Karin Tarte shows that the occurrence of aneuploidy in cultivated MSCs is not associated with the culture process and could be donor-dependent. All MSCs, with or without aneuploidy, become more senescent without exhibiting transformation features. Hence, karyotyping and fluorescent in situ hybridization (FISH) results are not informative and thus, are not adequate controls for the release of MSCs for clinical uses [56]. Our goal is to ensure that regulatory authorities, clinicians, and patients have access to the relevant risk/benefit ratios to help make an informed decision in the context of available treatments and technologies.

Challenges

The WJMSCs have displayed numerous advantages in preclinical trials. However, given that WJMSC transplantation has not yet been approved by the US Food and Drug Administration (FDA) and that clinical trials of MSC transplantation showing no adverse events have not yet reached the 10-year mark, WJMSCs have not been extensively used for clinical therapies. In addition, a number of challenges and side effects in WJMSC-based therapy remain to be addressed. First, the umbilical cord is an allogeneic tissue for the recipient; thus, transplantation needs to be crossmatched because of the HLA. The second problem is an ethical one. Although the umbilical cord is a disused organ, both the donor and the recipient have the right of informed consent. Third, a complete detection system to test any potential disease risk in donors has yet to be established. Fourth, the isolation and further expansion of primitive MSCs probably have several uncertain factors, such as the use of animal serum and the most common culture method in vitro, which should be avoided in clinical-grade therapy to prevent cell contact with xenogeneic proteins. Fifth, potential long-term risks associated with MSC therapy that may not have been observable within a short period after administrations have recently been confirmed in preclinical studies. These risks include potential maldifferentiation, immunosuppression, and malignant tumor growth, which is the primary safety concern, as well as the promotion of tumor growth when the treatment was systemically administered into animals with existing malignancy [57,58]. Lastly, as products of cell therapy, product consistency, cell stability, and toxicity should also be considered.

Prospect and future direction

Stem cell therapy is one of the most promising areas of regenerative medicine, and a number of such therapies are now under development by academic and industrial groups. As the technology of cell therapies has advanced, regulatory systems to monitor their use must be established. The general strategy for many adult stem cells and all pluripotent stem cell therapies is the scale up of undifferentiated stem cell production. After differentiation into a specific cell type and delivery to patients, the cells may reside indefinitely. Given that the signal pathway for MSC recruitment and repair is not clear, future studies need to be conducted to strengthen the mechanism and establish a series of criteria for the donor and the recipient. A good manufacturing practice (GMP) culture system for cell products during manufacture is essential in clinical therapy. Furthermore, MSC banking, which can support the successful establishment of umbilical cord blood banking, should be set up. Although the aforementioned challenges must still be addressed, the potential of WJMSCs in skin regenerative clinical treatments is promising.

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